There is no evident correlation between interleukin-10 gene polymorphisms and periportal fi brosis regression after specifi c treatment

Introduction: We evaluated the associations between interleukin-10 (IL-10) gene polymorphisms -G1082A/-C819T/-C592A and periportal fi brosis regression after specifi c treatment for schistosomiasis. Methods: This retrospective cohort study involved 125 Brazilian patients infected with Schistosomiasis mansoni, who were followed up for 2 years after specifi c treatment to estimate the probability of periportal fi brosis regression. Results: There was no evidence of associations between IL-10 polymorphisms and periportal fi brosis regression after treatment. Conclusions: There was no evidence of associations between gene promoter polymorphisms of IL-10 and the regression of periportal fi brosis in this Brazilian population.

Schistosomiasis is a chronic parasitic infection caused by the trematode Schistosoma, which affects about 240 million people worldwide (1) .Brazil is notable in the worldwide context of schistosomiasis because about 25 million Brazilians live in areas that present risks of contamination and about 4 to 6 million Brazilians are infected with the Schistosoma mansoni species.The Northeastern region of Brazil is particularly notable because of the high endemicity of schistosomiasis, including in the State of Pernambuco, which has the third-greatest prevalence of this disease in the country, as well as the highest number of deaths recorded for this disease (1) .
Schistosomiasis causes periportal fi brosis (PPF).The PPF process can result in the development of portal hypertension (PH), which itself leads to splenomegaly and the appearance of varicose veins in the esophagus.The rupture of these esophageal varices with consequent upper gastrointestinal bleeding (UGB) occurs in 12-15% of patients with the hepatosplenic form of the disease (2) .
The development of PPF is dependent on various immunogenetic and environmental factors.Sex, age, parasite load, alcohol consumption, exposure frequency, exposure duration, and other factors may affect the development and severity of PPF (3) .On the other hand, specifi c treatment may also contribute to reducing infection levels and consequently improve the clinical picture of hepatosplenomegaly and PPF (4) (5) .
In previous studies, it has been observed that the PPF process depends on the action of cytokines, since interleukin-10 (IL-10) has a central role in this regulation (2) (6) .The IL-10 secreted by Th2 cells is an important anti-infl ammatory, antifi brotic cytokine (6) .However, the role of IL-10 in the activation and inhibition of PPF in schistosomiasis is not well understood (7) .
The IL-10 gene contains fi ve sequences of nitrogenous bases responsible for protein-coding (exons) on the chromosome that is located in the positions between 1q31 and 1q32 (8) .The probable effects on gene transcription and protein production stimulated studies on polymorphisms in the promoter region of this gene, including -G1082A (rs1800870), -C819T IL-10 (rs1800871), and -C592A IL-10 (rs1800872) (8) .The thymine (T) and cytosine (C) alleles at position -819 in the promoter region of the IL-10 gene are in linkage disequilibrium with the adenine (A) and C alleles at position -592, respectively (8) .
Thus, this study was undertaken to investigate the associations of clinical factors (sex, UGB, specifi c treatment, and contact with focus) and IL-10 polymorphisms -G1082A/-C819T/-C592A with PPF regression after specifi c treatment in patients with S. mansoni.We specifi cally sought to investigate these associations in populations of endemic areas of the State of Pernambuco, Northeastern Brazil.
The study was performed retrospectively using a cohort obtained from a previous study of 203 patients who had schistosomiasis and received specifi c treatment for this disease.Additionally, -G1082A/-C819T/-C592A IL-10 polymorphisms and their respective serum concentrations determined for these patients (9) .The present cohort study included 119 patients infected with S. mansoni who were recruited during 2012-2013 and followed-up retrospectively for 2 years after specifi c treatment for S. mansoni to estimate the probability of PPF regression.Sociodemographic and clinical factors were also identifi ed, with an emphasis on specifi c treatment for S. mansoni.
In the previous study (9) with 203 patients, polymorphisms -G1082A/-C819T/-C592A IL-10 in the promoter region of the IL-10 gene were determined alongside their respective serum concentrations.Of the 203 patients in this previous study, the current study excluded 12 who had image pattern A (without PPF) in the fi rst abdominal ultrasound, 66 who did not have a follow-up of 2 years after the fi rst specifi c treatment for S. mansoni, and 6 for whom the genotype frequencies for regions (-C819T/-C592A) of the IL-10 gene were not determined.The fi nal analysis included 119 patients for these regions and 125 patients for the remaining region (-G1082A).
All patients were over 18 years of age and treated in the Gastroenterology Clinic of the Hospital das Clínicas, Federal University of Pernambuco (HC/UFPE; Recife, Brazil).Each patient was from an area of the State of Pernambuco in which schistosomiasis was endemic.
The study only included patients who had undergone abdominal ultrasound with PPF evaluation following the protocols of Niamey (10) and Cairo (11) , both before treatment for schistosomiasis and 2 years after treatment.We excluded patients with a history of other joint hepatic comorbidities, including fatty liver disease, liver cirrhosis, and hepatitis B or C, as well as patients with other clinical forms of schistosomiasis.Patients with hepatitis B or C were excluded based on serology for hepatitis B surface antigen, hepatitis B core antibody, and hepatitis C antibody.
The outcome of interest was PPF regression identifi ed by ultrasonography of the upper abdomen.The main exposures were polymorphisms -G1082A/-C819T/-C592A IL-10 which, in the context of the hypothesis of this study, were investigated for associations with the severity of PPF.The information on these genotypes and their respective serum IL-10 concentrations were obtained in 2012 and 2013 (9) .
To determine the genotypes (-1082) GA/AA and GG IL-10, (819T/-C592) CT/AC, TT/AA, and CC/CC IL-10, biological samples from all patients were previously submitted to polymerase chain reaction by specifi c allele, to detect the single base polymorphism in the region promoting the IL-10 gene (8) and thereby defi ne the exposure groups mentioned above.Serum levels of IL-10 were measured using a commercial enzyme-linked immunosorbent assay (ELISA) (Biosource; Invitrogen Corporation, Carlsbad, CA, USA), according to the manufacturer's instructions.The results were expressed in pg/ml based on the standard curves (sensitivity <1.0pg/mL).
The other investigated explanatory variables were sex, age, UGB, and contact with contaminated water.Data were collected using a precoded structured questionnaire that was applied to individuals by a single investigator.
The follow-up period was from the moment the specifi c treatment was fi rst recorded until 2 years afterwards.Relative risks (RRs) and 95% confi dence interval (95% CIs) were estimated in bivariate analyses.The signifi cance level was set at 0.05.Epi Info version 3.5.5 (Centers for Disease Control, Atlanta, GA, USA) was used for all statistical analyses.The Kruskal-Wallis test was used to compare the serum levels of IL-10 between groups.
Overall, the average age of patients was 57 years (standard deviation, 13 years).The frequency distributions of the sociodemographic and clinical variables are summarized in Tables 1 and 2, comparing the exposure groups for polymorphisms (-C819T/-C592A) and (-G1082A), respectively.The (-C819T/-C592A) polymorphism groups did not differ signifi cantly for any of the investigated factors (Table 1).However, as shown in Table 2, women had polymorphism -G1082A more commonly than men (p = 0.044).
As has already been established in the literature, several environmental factors influence the natural history of PPF, including age, exposure frequency, parasite load, specifi c treatment,  and level of education (3) (7) (12) .Among the investigated patients who were infected with S. mansoni, there was no statistically significant difference in any of these factors between the exposure groups.This suggests that there is a need for further cohort studies to assess other risk factors that have not been addressed in the present study, but that might explain the environmental risk factors in the investigated population.
The immune response regulated by the genetics of the host is believed to play a central role in the natural history of PPF.In this study, which involved Brazilian patients infected with S. mansoni, no signifi cant association was found between polymorphisms -G1082A/-C819T/-C592A IL-10 and PPF regression.Additionally, there was no difference in serum levels of IL-10 between exposure groups.Recently, Silva et al. (9) evaluated the effects of these polymorphisms on the severity of PPF in 203 patients infected with S. mansoni who had different patterns of PPF in the State of Pernambuco.Silva et al. found that only polymorphism (-G1082A) showed risk associations with disease severity in this Brazilian population.The authors concluded that these results may have been infl uenced by ethnic variations.Silva et al. also found no difference between the mean serum levels of IL-10 between clinical groups.However, in a study of 812 patients with schistosomiasis in an endemic region in Brazil, Grant et al. (13) found a signifi cant association between polymorphism (-T819C) IL-10 and the nonspecifi c immune response, but not with the ovular immune response to S. mansoni.Accordingly, these results were not associated with the development of PPF.
Controversial results have been observed in the majority of studies that have compared genotype distributions of polymorphisms of the IL-10 gene promoter with fibrosis severity (12) (14) (15) .Jin et al. (14) investigated the role of IL-10-592 A/C, IL-10-819 C/T, and IL-10-1082 A/G gene polymorphisms in the development of liver cirrhosis in Asian populations, demonstrating that the IL-10-592 A/C gene polymorphism enhances the risk of liver cirrhosis, and that this gene variant interacts with chronic hepatitis B infection in this population.
Guo et al. (12) analyzed 197 moderate/severe liver fi brosis cases and 426 mild fi brosis controls, as well as 536 cases of liver cirrhosis and 881 non-cirrhosis controls and concluded that the IL10-819 T allele may be a susceptibility factor for liver cirrhosis related to hepatitis C virus in the Japanese population.
On the other hand, Sofi an et al. (15) studied 96 patients infected with the hepatitis B virus (HBV) from a reference center for hepatitis in Central Iran, as well as and 31 healthy controls from a blood transfusion organization.The authors sought to evaluate the correlation between HBV and polymorphism (-G1082A) IL-10.They concluded that these genotypes were not signifi cantly different among the controls, patients who had recuperated from HBV, and patients who were carriers of or had chronic HBV.Thus, they found signifi cant corrections with neither the prognosis of infection with HBV, nor consequent hepatic fi brosis.These results could be attributable to the infl uence of other genes in the progression of HBV, and are not necessarily solely attributable to a direct relationship between the expression of IL-10 and the outcome of infection by HBV.
The results found in the present study do not exclude the possibility that other cytokines and genes infl uence PPF regression.Therefore, future studies with larger samples are needed to fully analyze these polymorphisms and their respective serum levels of IL-10 serum, to better assess whether there are connections between -G1082A/C819T/-C592A IL-10 polymorphisms, the expression of IL-10, and PPF intensity.If present, such associations may infl uence PPF regression after specifi c treatment for schistosomiasis.
In summary, there was no evidence of associations between gene promoter polymorphisms of IL-10 and the regression of PPF in this Brazilian population.Considering the limitations imposed by the sample size of the present study, further studies are recommended to explore the associations between these polymorphisms and the regression of PPF.

Ethical considerations
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the CH/UFPE (CAAE: 03161512.6.0000.5208).

TABLE 1
Distribution of sociodemographic and clinical variables according to polymorphisms (-C819T/-C592A) IL-10 in patients with schistosomiasis, State of Pernambuco, Brazil.

TABLE 2
Distribution of sociodemographic and clinical variables according to polymorphism (-G1082A) IL-10 in patients with schistosomiasis.State of Pernambuco, Brazil.

TABLE 3
Bivariate analyses of the associations between sociodemographic variable, clinical variables, polymorphisms (-C819T/-C592A) IL-10, and the regression pattern of periportal fi brosis in patients with schistosomiasis State of Pernambuco, Brazil.