Advanced periductal fibrosis from infection with the carcinogenic human liver fluke Opisthorchis viverrini correlates with elevated levels of interleukin-6

Authors


  • Potential conflict of interest: Nothing to report.

  • The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Disease or the National Institutes of Health.

Abstract

More than 750 million people are at risk of infection with foodborne liver flukes. Opisthorchis viverrini is considered among the most important of these parasites, due to its strong association with cholangiocarcinoma (CCA). O. viverrini infection results in a chronic inflammatory challenge to the host, which can lead to advanced, pathogen-specific disease sequelae including obstructive jaundice, hepatomegaly, cholecystitis, as well as CCA. However, before disease sequelae are apparent, important inflammatory changes to the liver can be detected early during O. viverrini infection. In a case-control study involving 328 men and women with O. viverrini infection, we determined the presence of advanced periductal fibrosis in asymptomatic, O. viverrini-infected individuals and then measured cytokine responses to O. viverrini excretory/secretory products (ES). In the 200 participants with advanced periductal fibrosis (cases), levels of interleukin-6 (IL-6) to O. viverrini ES were 8 times higher than levels of the 128 O. viverrini-infected individuals without advanced periductal fibrosis (controls). Moreover, elevated IL-6 to parasite ES was associated with increased risk of advanced periductal fibrosis by 63% in a model adjusted for sex and age. The risk of advanced periductal fibrosis was also found to increase with higher levels of IL-6: individuals in the third quartile of IL-6-ES production had a 127% higher risk of developing advanced periductal fibrosis than individuals in the first quartile of IL-6 production. O. viverrini-infected individuals with advanced periductal fibrosis showed other hepatobiliary abnormalities, including reduced gallbladder contractility and the presence of gallbladder sludge. Conclusion: These data strongly implicate a role for parasite-specific IL-6 in the pathogenesis of advanced periductal fibrosis in opisthorchiasis, with possible links to other hepatobiliary abnormalities, including CCA. (HEPATOLOGY 2009.)

Foodborne trematodiases represent an important group of communicable diseases, and one of the most clinically significant infectious pathogens behind malaria, tuberculosis, and human immunodeficiency virus (HIV).1, 2 At least 750 million people (10% of the world's population) are at risk of foodborne trematodiases, with more than 40 million people currently infected.1, 3Opisthorchis viverrini is considered among the most important of the foodborne trematodes due to its strong association with liver disease4 and cholangiocarcinoma (CCA).5, 6 Humans become infected with O. viverrini by consuming raw or undercooked fish that contain the infective stage. The parasites migrate up the biliary tract, establish a chronic infection (on average 4-5 years) in the intrahepatic bile ducts, and produce eggs that are excreted in the feces. Although the infection is effectively eliminated by the antihelminthic praziquantel, environmental and cultural factors of East Asia strongly favor the process of reinfection.7, 8

Opisthorchis viverrini infection represents a chronic inflammatory challenge to the host. The sustained production of growth factors and fibrogenic cytokines in response to local mechanical, toxic, and immune irritation results in a persistent inflammatory condition in the liver that progressively remodels and destroys the normal tissue architecture of the biliary epithelium.3, 9, 10 The cumulative damage caused by this chronic inflammation can lead to advanced, pathogen-specific disease sequelae including obstructive jaundice, hepatomegaly, cholecystitis, and CCA.4, 11 Whereas these are the acknowledged signs of morbidity in opisthorchiasis, important inflammatory changes to the liver can occur early in the course of the disease,12, 13 most of which will remain clinically silent unless actively detected by ultrasound or other imaging modalities.12, 14 Previous community-based ultrasound studies in O. viverrini endemic areas of northeastern Thailand suggest that hepatobiliary abnormalities such as enlargement of the left hepatic lobe and the gallbladder, loss of gallbladder contractility, presence of sludge, and increased periportal fibrosis are common.14, 15 Due to the high prevalence of O. viverrini infection in East Asia, which can be as high 60%-70% in populations resident in endemic areas,11 asymptomatic hepatobiliary abnormalities may represent the greatest part of the disease burden associated with opisthorchiasis. Multiple risk factors are likely to determine whether the host develops advanced periductal fibrosis from O. viverrini infection, including the duration of infection,15 the intensity of the infection,14, 15 and diet (nitrosamines).16 However, the risk factors that induce periductal fibrosis are likely to pass through the proinflammatory cytokine network, which includes transforming growth factor-β, interleukin (IL)-1-α, and IL-6.17

IL-6 is a pleiotropic cytokine with a broad range of humoral and cellular immune effects relating to inflammation, host defense, and tissue injury.18 It is of particular importance in diseases where the pathology arises from chronic inflammation (see Refs.17 and19 for review). Although crucial for the quick induction of an innate immune response,19 the persistent production of IL-6 may be central to the state of chronic inflammation induced during O. viverrini infection. Elevated levels of IL-6 have been reported in almost every chronic inflammatory disease of the liver,17 including alcoholic hepatitis20 and hepatitis B (HBV) and hepatitis C (HCV) viruses.21–26 More important, IL-6 appears to be a pivotal cytokine for cholangiocarcinogenesis. In chronically inflamed biliary epithelium (e.g., during O. viverrini infection), epithelial cells are constantly stimulated to participate in the inflammation by continuously secreting chemokines and cytokines,3, 4 creating a cellular microenvironment beneficial to cancer growth. In this regard, IL-6 appears to be a pivotal cytokine for both inflammation and cholangiocarcinogenesis.17, 19

On the basis of this background information, we sought to determine whether levels of proinflammatory cytokines are elevated among O. viverrini-infected individuals with advanced periductal fibrosis. We further sought to determine whether the association between markers of chronic inflammation and their association with advanced periductal fibrosis were modified by other factors including age, sex, and the intensity of O. viverrini infection.

Abbreviations

ALT, alanine aminotransferase; ALP, alkaline phosphatase; ANCOVA, analysis of covariance; AST, aspartate aminotransferase; CCA, cholangiocarcinoma; CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; epg, eggs per gram of feces; ES, excretory/secretory; HBA, hepatobiliary abnormalities; Ig, immunoglobulin; IL, interleukin; INF, interferon; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor; US, ultrasonography.

Patients and Methods

Study Design and Setting.

The current study represents an analysis of baseline data collected from a community-based, case-control study of the risk factors associated with the development of advanced periductal fibrosis in opisthorchiasis. Individuals from seven villages in the vicinity of the regional capital, Khon Kaen, Khon Kaen Province, Thailand, were surveyed27 (Supporting Fig. 1). For the current study, 1032 individuals were screened for O. viverrini infection, with 554 positive (53.7%) for O. viverrini infection. From this group of infected individuals, 200 males and females between the ages of 20 and 60 years old with advanced periductal fibrosis were enrolled as “cases” and asked to provide 30 mL of whole blood for baseline immunology and hematological parameters. Individuals who were positive for O. viverrini but negative for advanced fibrosis were defined as “controls” and matched with cases based on geographic location of residence (nearest-neighbor). As such, 128 individuals were identified as controls and were asked to provide 30 mL of blood for baseline immunology and hematologic parameters. All individuals (cases and controls) positive for O. viverrini were referred to the local public health outpost for treatment with praziquantel (standard of care). This study was approved by the Ethics Committee of Khon Kaen University School of Medicine, Khon Kaen, Thailand (reference number HE480528) and the Institutional Review Board of the George Washington University School of Medicine, Washington, DC (GWUMC IRB# 020864).

Figure 1.

Representative ultrasonographs, revealing increasing periportal echoes and markedly enlarged gallbladder, in individuals categorized as grade 0 (A), 1+ (B), 2+ (C), and 3+ (D) periportal fibrosis. Individuals were classified as periductal fibrosis grade 0 when no echoes were observed (A); 1+ when echoes were observed in one segment of the liver (B); 2+ when echoes were observed in two or three segments of the liver (C); and, 3+ when echoes were observed in greater than three segments of the liver (D). Individuals were then dichotomized into “nonadvanced fibrosis” or “control” if the US grade was 0 or 1 and “advanced fibrosis” or “case” if the US grade was 2 or 3.

Ultrasonography.

A mobile, high-resolution ultrasound (US) machine (GE model LOGIQ Book XP) was used. Hepatobiliary abnormalities (HBA) including portal vein radical echoes, echoes in liver parenchyma, indistinct gallbladder wall, gallbladder size, sludge, and suspected CCA were graded and recorded as described.28, 29 For the study of gallbladder function (contractility), the gallbladder was measured before and then 30 minutes after consumption of a fatty meal.28 Individuals were classified as periductal fibrosis grade 0 when no echoes were observed in any segment of the liver (Fig. 1A); 1+ when echoes were observed in one segment of the liver (Fig. 1B); 2+ when echoes were observed in two or three segments of the liver (Fig. 1C); and, 3+ when echoes were observed in greater than three segments of the liver (Fig. 1D). Individuals were then dichotomized into cases and possible controls as follows: “Non-Advanced Fibrosis” or “control” if the US grade was 0 or 1, and “Advanced Fibrosis” or “case” if the US grade was 2 or 3. Two radiologists performed US readings over the course of the study. A weighted kappa score was used to quantify level of agreement between the two readers.30 If a kappa fell below 0.6, a third ultrasonographer was consulted to read the image and determine the final outcome.

Fecal Exams.

Two grams of feces were weighed and preserved in 10 mL of 10% formalin in a 15-mL screw-cap centrifuge tube, labeled, mixed, and kept at room temperature until processing for quantitative formalin/ethyl acetate concentration technique.15 The presence and intensity of O. viverrini infection was determined by quantitative formalin/ethyl acetate technique and expressed as eggs per gram of feces (epg). Eggs/parasite identification and egg counts were under light microscopy at 10× and 40× magnifications, with the results reported as the number of epg.

Urine Pregnancy Testing.

β-hCG testing was performed at the study site using urine pregnancy test kits approved by Thai and/or US regulatory agencies.

Parasite Antigen Preparation.

Excretory/secretory antigen (ES) of cultured O. viverrini adult worms obtained from experimentally infected hamsters was prepared as described.31 In order to eliminate any possible stimulatory effects in tissue culture from bacterial lipopolysaccharide (LPS) in crude O. viverrini antigen preparations, LPS was removed from the crude antigen extract by phase separation using Triton X-114.32 These studies were approved by the Animal Ethics Committee of Khon Kaen University (#0514.1.12.2/23) and the Institutional Animal Care and Use Committee, George Washington School of Medicine.

Blood Collection by Venipuncture.

Approximately 2 mL of blood was collected in EDTA-coated tubes for hematology and ≈20 mL of whole peripheral blood was obtained in heparinized tubes for cellular immunology and cytokines assays.

Liver Functions Tests.

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) assays in serum samples from cases and controls were performed on an automated Synchron CX-4 system (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions.

Enzyme-Linked Immunosorbent Assay.

O. viverrini-specific immunoglobulin G (IgG), IgG1, IgG2, IgG3, and IgG4 were determined by indirect enzyme-linked immunosorbent assay (ELISA) and parasite-specific IgE was examined by indirect avidin-biotin ELISA as described in Sripa and Kaewkes.31

Cellular Immune Response and Cytokine Measurements.

Cytokine production by peripheral blood mononuclear cells (PBMCs) in the presence of O. viverrini ES were examined at 0, 48, and 72 hours for levels of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, interferon (INF) γ, tumor necrosis factor (TNFα), and TNFβ production. A whole blood culture technique was used for cytokine stimulation. Appropriately diluted (1:16) heparinized whole blood was cultured in RPMI 1640 medium in 24-well tissue culture plates in the presence of 35 μg/mL of O. viverrini ES crude antigen extract or 2.5 μg/mL for phytohemagglutinin (PHA)-L (Difco Laboratories, Detroit, MI). Cells were cultured at 37°C in a humidified 5% CO2 incubator. Supernatants were removed at predetermined times (when maximum secretion of each cytokine was obtained between 48-120 hours of culture) and stored in aliquots at −80°C. Twenty-five to 50 μL of supernatant was used for all cytokine assays. The quantification of secreted cytokines was analyzed using commercial FlowCytomix bead-based multiplexing assays kits (Beckman-Coulter). Values were quantified from standard curves using human recombinant cytokines.

Statistical Analyses.

Cases and controls were compared for dichotomous and categorical variables by percent and then differences tested with Fisher's Exact test. In the case of continuous variables, two methods were used to assess for statistical significance. When continuous variables were normally distributed, Student's t test was used to determine differences between cases and controls. In the special case of gallbladder contractility, where the difference in gallbladder dimensions between pre- and post-fatty meal were the variable of interest, an analysis of covariance (ANCOVA) was used to test for differences between cases and controls. When the data were (1) highly skewed, (2) had a number of outlier values, or (3) were refractory to transformation (e.g., log-transformation), a quantile regression model was used to determine the median along with 95% confidence intervals (95% CI) to estimate the differences between cases and controls: e.g., cytokine level, liver function tests, and antibody levels. Where the data suggested a significant difference between cases and controls, such as in the levels of IL-6 to O. viverrini ES, the risk of this parameter on advanced periductal fibrosis was assessed using a logistic regression model. Odds ratios (OR) were estimated on the risk of advanced periductal fibrosis in crude models and models adjusted for age and sex. In the case of IL-6 production to O. viverrini ES, the variable was treated in its original scale, a categorical scale (using the median value as a cutoff), and an ordinal scale determined by quartiles of IL-6 production to ES. Stata v. 10 (StataCorp, College Station, TX) statistical software was used for all analyses. Level of significance in statistical tests was 0.05. All tests were two-tailed.

Results

Study Design.

The baseline characteristics of the enrolled participants are shown in Table 1, with 200 cases and 128 controls enrolled in the study. Overall, the sample included slightly more females (52%) than males (48%) (P = 0.083). The study sample had a mean age of 45.8 and an age range of 20 to 60 years, with 74% of the study sample over 40 years of age. The mean ages for cases (46.7 years of age) and controls (45.2 years of age) did not differ significantly (P = 0.115). As part of the inclusion criteria of the study, both cases and controls were positive for O. viverrini as determined by a single ovum in feces. No statistically significant difference (P > 0.811) was observed in the intensity of O. viverrini infection between cases (median = 36 epg) and controls (median = 38 epg). As shown in Table 1, in both case and control groups there was a similar distribution of the proportion of individuals when intensity of O. viverrini infection was stratified by tertiles of epg, with the majority of cases (77%) and controls (77%) having less than 499 epg.

Table 1. Relationship Between Advanced Periductal Fibrosis Status as Determined by Ultrasonography in Opisthorchis viverrini-Infected Individuals by Age, Sex, and Level of Infection
 Advanced Periductal Fibrosis
Negative (0 and 1)Positive (2 and 3)
  1. Epg, eggs per gram of feces.

Total128200
   
Sex  
 Male54 (42.2%)104 (52.0%)
 Female74 (57.8%)96 (48.0%)
   
Age (years)  
 20-294 (3.1%)17 (8.5%)
 30-3921 (16.4%)42 (21.0%)
 40-4956 (43.8%)75 (37.5%)
 50+47 (36.7%)66 (33.0%)
   
Intensity of infection  
 1-499 epg98 (76.6%)154 (77.0%)
 500-999 epg16 (12.5%)18 (9.0%)
 >1000 epg14 (11.0%)28 (14.0%)

Individuals with Advanced Periductal Fibrosis Show Poor Gallbladder Function.

Table 2 shows that the gallbladder did not contract as much after a fatty meal in individuals with advanced periductal fibrosis compared to individuals without advanced periductal fibrosis. In addition, individuals with advanced periductal fibrosis had a greater presentation of sludge than individuals without advanced periductal fibrosis for whom sludge was entirely absent. These data indicate that individuals with advanced periductal fibrosis from O. viverrini have reduced gallbladder function.

Table 2. Relationship Between Gall Bladder Dimensions “Pre” and “Post” Fatty Meal and Sludge with the Presence of Advanced Periductal Fibrosis by Case and Control Status
Gall Bladder AbnormalitiesAdvanced Periductal FibrosisP
Negative Mean (SD)Positive Mean (SD)
  • *

    The difference in gall bladder dimensions (measured in centimeters) “pre” and “post” fatty meal.

Post-fatty meal reduction*   
 Length1.88 (1.35)1.59 (1.20)0.007
 Width0.71 (0.55)0.68 (0.56)0.154
 Cross-sectional0.61 (0.47)0.55 (0.57)0.261
    
 n (%)n (%) 
Presence of sludge013 (6.5)0.002

Advanced Periductal Fibrosis in Opisthorchiasis Does Not Appear to Affect Liver Function.

There was no difference in the levels of the liver function tests ALT, AST, and ALP between cases and controls (Supporting Table 1), indicating that pathology may be limited to advanced periductal fibrosis and did not affect liver function.

Intensity of Infection Was Not a Risk Factor for Advanced Periductal Fibrosis in O. viverrini Infection.

Table 1 shows the individuals by the intensity of O. viverrini infection: “≤499 epg” (77%), “500-999 epg” (10%), and “≥1,000 epg” (13%). No significant difference was found (P = 0.696) between the median epg for individuals with advanced periductal fibrosis (median, 141 epg) compared to controls (median, 121 epg). Table 3 shows that intensity of infection (expressed as epg) was not a risk factor for advanced periductal fibrosis. When epg was measured at an interval of 1 epg, the odds ratio was 0.99 (95% CI 0.99-1.00, P = 0.811) and when measured in the larger interval of 500 epg the odds ratio remained unchanged 0.99 (95% CI 0.93-1.00, P = 0.811). The relationship between baseline levels of epg and advanced periductal fibrosis was not altered by analyses that adjusted for age, sex, or age and sex simultaneously (Table 3).

Table 3. Crude and Adjusted Odds Ratios for Intensity of Infection, Expressed as Eggs per Gram of Feces (epg) for Opisthorchis viverrini Infection and Advanced Periductal Fibrosis
 Odds Ratio95% CIP
  1. CI, confidence interval.

Intervals of 1 epg   
Crude0.990.99, 1.000.811
Adjusted   
 Age0.990.99, 1.000.987
 Sex0.990.99, 1.000.646
 Age and sex0.990.99, 1.000.818
Intervals of 500 epg   
Crude0.990.93, 1.000.811
Adjusted   
 Age0.990.94, 1.060.987
 Sex0.990.93, 1.050.646
 Age and sex0.990.93, 1.060.818

Levels of IL-6 to O. viverrini ES Were Significantly Higher in Individuals with Advanced Periductal Fibrosis.

Of the 11 cytokines tested (Supporting Table 2), only levels of the inflammatory cytokine IL-6 were significantly elevated in individuals with advanced periductal fibrosis (Table 4). The frequency distributions of baseline IL-6 levels to O. viverrini ES are presented for cases and controls combined in Fig. 2A, for cases only in Fig. 2B, and for controls only in Fig. 2C. IL-6 production from PBMCs to O. viverrini ES among both cases and controls ranged between −2.7791 and 2.3082 pg/mL, with a range much greater in controls (−27,791 to 23,082 pg/mL) than in cases (−116.4 pg pg/mL to 14,221 pg/mL), indicating far less variation in the IL-6 response to O. viverrini ES among cases than controls. Median baseline levels of IL-6 in the presence of O. viverrini ES were 8 times higher in PBMCs from cases than controls (3981 versus 507 pg/mL; P = 0.001). Individuals with “<499 epg” showed 12 times higher levels of IL-6 than controls (4,305.9 versus 348.9 pg per mL; 95% CI 1,500.9 to 5,568.6; P = 0.001).

Table 4. Baseline Concentrations (Median) of IL-6 in Supernatants from Peripheral Blood Mononuclear Cells (PBMC) Stimulated with Opisthorchis viverrini Excretory/Secretory Products After 48 Hours According to Case (Advanced Periportal Fibrosis Positive) and Control (Advanced Periportal Fibrosis Negative) Status and in Relationship to Sex, Age, and Intensity of Infection
NUnstimulated PBMCStimulated PBMCIL-6 Production by PBMC
Control 128Case 200PControl 128Case 200PControl 128Case 200P
  1. Production refers to the median of the differences between stimulated PBMC and unstimulated PBMC (control wells) as expressed by the equation: Production = Median (Stimulated PBMC– Unstimulated PBMC).

Total999.21408.90.4784478.68415.30.145507.13981.20.001
          
Sex         
 Male1842.31288.00.501908.37436.40.0164.12772.60.039
 Female739.01506.40.1388032.110440.60.6801404.74966.20.082
          
Age (years)         
 20-292452.4421.60.03619.721.50.999-154.72.50.963
 30-39404.42540.50.0188262.33729.60.530495.91298.60.344
 40-491872.4104.60.143636.08790.80.0053.23648.20.014
 50-59820.91920.40.0308604.99217.90.8731687.86030.00.116
          
Intensity of infection (epg)         
 <499959.51532.00.3184286.78603.00.113348.94305.9<0.001
 500-9993645.7280.5<0.0013817.53918.10.9392135.93473.40.333
 1000-20002877.22572.20.98412694.49077.30.9919017.32870.90.524
Figure 2.

The frequency and cumulative percentage of production of IL-6 to Opisthorchis viverrini ES products among cases and controls in a community-based case control study. The bars refer to frequency of production of IL-6 and the line refers to cumulative frequency of IL-6. (A) All individuals in the study (n = 328); (B) only to cases (n = 200); (C) only to controls (n = 128).

Elevated Levels of IL-6 to O. viverrini ES Significantly Increase the Risk of Developing Advanced Periductal Fibrosis.

Table 5 shows that elevated levels of IL-6 to O. viverrini ES at baseline significantly increased the risk (OR = 1.63) of advanced periductal fibrosis in a model adjusted for age and sex (95% CI 1.01 to 2.54, P = 0.048). Moreover, as shown in Table 6, the risk of advanced periductal fibrosis increased with increasing quartiles of IL-6 production to O. viverrini ES: OR = 1.00 for Quartile 1 (reference quartile); OR = 1.39 for Quartile 2; OR = 2.27 for Quartile 3; and OR = 1.64 for Quartile 4. However, only for individuals in Quartile 3 of IL-6 production to O. viverrini ES was the risk (OR = 2.27) statistically significant for advanced periductal fibrosis (95% CI = 1.18 to 4.368, P = 0.014).

Table 5. Crude and Adjusted Odds Ratios for Levels of IL-6 by Peripheral Blood Mononuclear Cells (PBMC) Stimulated by Opisthorchis viverrini Excretory/Secretory Products (ES) for Advanced Periductal Fibrosis*
 Odds Ratio95% CIP
  • *

    *In vitro cultures of PBMC with O. viverrini ES after 48 hours. CI, confidence interval.

Crude1.510.94, 2.410.089
Adjusted   
 Age1.560.97, 2.510.066
 Sex1.550.96, 2.500.070
 Age and sex1.631.01, 2.540.048
Table 6. Crude and Adjusted Odds Ratios by Quartiles of the Difference Between IL-6 Produced by PBMC That Were Unstimulated or Stimulated by Opisthorchis viverrini Excretory/Secretory Products (ES) for Advanced Periductal Fibrosis Among O. viverrini-Infected Individuals
 Quartile IL-6 in Production to O. viverrini ES (range, pg/mL)P for Trend
1 (–47,073.0 to–2.44)2 (–2.45 to 2695.8)3 (2695.9 to 12,519.1)4 (12,519.2 to 44,055.3)
  1. CI, confidence interval; NA, not applicable to adjusted models; OR, odds ratio. Adjusted models include age and sex simultaneously.

Crude     
 OR1.001.352.071.49P = 0.106
 95% CI0.73-2.501.09-3.920.80-2.78 
 P0.3460.0260.207 
Adjusted     
 OR1.001.382.271.64NA
 95% CI0.74-2.591.18-4.380.87-3.08 
 P 0.3120.0140.127 

Antibody to O. viverrini ES Did Not Associate with Advanced Periductal Fibrosis.

There was no significant increase between cases and controls in levels of IgG and its subclasses (IgG1, IgG2, IgG3, and IgG4) or IgE to O. viverrini ES (Supporting Table 3). Although important for O. viverrini infection, these data indicate that humoral immunity may not be a risk factor for advanced fibrosis.

Discussion

Liver fluke infection with O. viverrini is associated with a number of inflammation-induced hepatobiliary pathologies, the most common of which is periductal fibrosis.28 To our knowledge, this is the first report of an association between an inflammatory cytokine and periductal fibrosis in liver fluke infection. IL-6 levels to O. viverrini ES were on average 8 times higher in O. viverrini-infected individuals with advanced periductal fibrosis compared to O. viverrini-infected individuals without periductal fibrosis. Moreover, baseline PBMC production of IL-6 to O. viverrini ES significantly elevated the risk (63%) of advanced periductal fibrosis in a model adjusted for age and sex, with the risk of advanced periductal fibrosis increasing with increasing IL-6 levels to parasite antigen. The current data strongly support our hypothesis that opisthorchiasis represents a chronic inflammatory disease mediated by proinflammatory cytokines, specifically IL-6, which is significantly associated with the development of advanced periductal fibrosis.

Previous community-based US studies in O. viverrini endemic areas of northeastern Thailand suggest that hepatobiliary abnormalities such as enlargement of the left hepatic lobe and the gallbladder, loss of gallbladder contractility, presence of sludge, and increased periductal fibrosis are common.15, 28, 34 In the current study we also document deficits in gallbladder function, specifically reduced gallbladder contractility and the presence of sludge in O. viverrini-infected individuals with advanced periductal fibrosis. Recent studies in animals34 and humans4 have shown that opisthorchiasis is associated with chronic cholecystitis, which is characterized by inflammatory cell infiltration and prominent fibrosis of the gallbladder wall. The chronic inflammation and fibrosis of the gallbladder wall14 can impair gallbladder contractility as observed in the current study among the individuals with advanced periductal fibrosis.14 The poor contractility of the gallbladder as well as the periductal fibrosis of the intrahepatic bile ducts may also cause functional bile stasis (or sludge). This can lead to precipitation of bile contents, which may even include O. viverrini eggs.35 Periportal and periductal fibrosis are among the most prominent histological features in chronic O. viverrini infection in humans36 and in long-term O. viverrini infection in hamsters.37 In a series of human autopsies conducted during the 1970s,38 no detectable changes were observed in the biliary epithelium and periductal areas of the liver in individuals with recent O. viverrini infection, whereas individuals with chronic O. viverrini infection had a proliferation of epithelial cells and the formation of acini and periductal fibrosis. Our observations support the hypothesis that, as with other chronic helminth infections,8 the asymptomatic abnormalities represent an important component of the disease burden in opisthorchiasis.

Chronic inflammatory disorders such as those seen in opisthorchiasis are induced by persistent irritants that sustain the production of growth factors and fibrogenic cytokines, which in turn stimulate the deposition of connective tissue elements that progressively remodel and destroy normal tissue architecture, resulting in fibrotic elements.10 In opisthorchiasis, the persistent irritation can come from one or both of the following sources: (1) the feeding and migration of flukes (mechanical irritation), when oral and ventral suckers hook onto the biliary epithelium and damage tissue or (2) metabolic products that are released from the tegument and excretory openings of the parasite and enter into the bile duct epithelium of the human host.4 From the current data, we hypothesize that the persistent irritant derives from some component(s) of the crude ES antigen preparation that we employed to stimulate PBMCs in vitro, which resulted in elevated IL-6 production. ES antigens have proven to be important immune modulators in a number of helminth infections,39 usually skewing the host cytokine response to create a microenvironment favorable to parasite survival. In the current study we offer a novel role for helminth ES products: the idea that ES products from O. viverrini may be toxic or may interact with the biliary epithelium as a mitogen36 that results in chronic inflammation and subsequent periductal fibrosis and possibly even CCA. This hypothesis is consistent with much of the experimental literature on O. viverrini ES products in laboratory models. For example, when O. viverrini adults worms were cocultured with human biliary cell lines in a noncontact transwell system, they induced a proliferative response,4 indicating that soluble products excreted or secreted from the parasite are indeed capable of stimulating cells to proliferate. A similar observation was also found when O. viverrini adult worms were cocultured with mouse NIH-3H3 fibroblasts.40 There is also evidence that these immunological responses are, at least in part, specifically evoked by O. viverrini products: in this same noncontact transwell system, T-cell-deprived hamsters did not show a proliferative response when cocultured with adult worms.41 Our study extends these findings by indicating that ES products in O. viverrini-infected humans may cause a persistent irritation, resulting in a chronic inflammatory state, mediated by IL-6, which is strongly associated with advanced periductal fibrosis.

IL-6 is known to play a role in a number of chronic inflammatory conditions of the liver, e.g., alcoholic and viral hepatitis42, 43 and other well-known fibrotic lesions such as keloid pathogenesis.44 One route by which IL-6 may be responsible for periductal fibrosis in opisthorchiasis is the up-regulation of profibrotic cytokines such as TGFβ1,10 which has been observed in experimental O. viverrini infection in hamsters.45, 46 In addition, IL-6 is a pleiotropic cytokine involved in a variety of inflammation associated cancers,17 including CCA.47 In particular, in chronically inflamed biliary epithelium, epithelial cells are constantly stimulated to participate in the inflammation by continuously secreting chemokines and cytokines, which establishes a cellular microenvironment that is conducive to neoplasia.48Opisthorchis may be the first helminth infection linking the development of fibrosis with parasite specific IL-6 production.

This study emphasizes the significant relationship between the inflammatory cytokine IL-6 and advanced periductal fibrosis in opisthorchiasis. Although less visible in clinical presentation, asymptomatic hepatobiliary abnormalities in opisthorchiasis may actually represent the greatest part of the chronic disease burden associated with this very prevalent but also neglected tropical disease.7, 8 Further studies are needed to enhance our knowledge of the relationship between proinflammatory cytokines, hepatobiliary pathology, and cholangiocarcinogenesis. Such studies will then allow us to identify additional risk factors and biomarkers for disease progression, highlighting those individuals with the highest risk of developing pathologic sequelae and CCA.

Acknowledgements

We thank the volunteers who participated in this study. We also thank our regulatory affairs and data management colleagues including Preeyaporn Plaimee, Sangduan Wannachart, Sombat Thinkhamrop, Wilaiporn Thinkhamrop, and Jittiawadee Pukdeepromma, and our laboratory and field technicians, including Arpa Surapitoon for assistance with the flow cytometric work. We thank Dr. Pyatat Tatsanavivat and Tarika Samor of the Clinical Research Collaboration Network, Thailand, for guidance with compliance issues.

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