IL-10 and TNF-α genes polymorphisms and the development of cervical lesions and cervical adenocarcinoma: a case-control study

Cytokines have an important role in the progression of cervical lesions and/or adenocarcinoma. We investigated whether polymorphisms at the promoter regions of the IL-10 -1082 (A> G, rs1800896) and TNF-α -308 (G>A, rs1800629) genes were associated with susceptibility to progression of cervical dysplasia and adenocarcinoma. The study consisted of 240 women infected with HPV (72 with adenocarcinoma and 168 with cervical intraepithelial lesions), and 169 healthy control women. There was a significant increase in the frequency of the IL10 -1082G allele in both cervical dysplasia (OR = 1.39; P = 0.0372) and adenocarcinoma patients (OR = 2.19; P = 0.0002). For the TNF-α -308 polymorphism, there was higher susceptibility to cervical lesions, in relation to risk factors such as: age > 35 years old (OR = 2.57; p = 0.0057), age of first sexual intercourse 1st < 18 years old (OR = 6.6224, p < 0.0001), smoking (OR = 3.80; P = 0.0003), African ancestry (OR=5.18, p < 0.0001) and co-infection with Chlamydia trachomatis (OR=2.41, p=0.0315). Our findings suggest that polymorphisms in the IL-10 and TNF-α genes may play a role in the susceptibility or severity of cervical disease in the study population.


INTRODUTION
Despite infection by one of the types of human papillomavirus of high oncogenic risk (HR-HPV) being recognized as the main cause of cervical cancer and its precursor lesions (Cervical Intraepithelial Neoplasms CIN), Ferlay et al.(2008) indicated that the presence of other cofactors plays an important role in viral etiology, such as: alcoholism, multiple partners, multiparity, use of oral contraceptives, age, and co-infections with other types of pathogens [Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) and Chlamydia trachomatis (CT)]. Furthermore, Moscicki et al. (2012) pointed out the need to also take into consideration genetic factors of the host. The type and quality of the immune response are factors that favor an enabling environment for viral replication, and individuals with impaired cellular immune response have a high prevalence of cervical lesions induced by HPV (Stanley, 2006).
Interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-α) are two multifunctional cytokines involved in the immune response of the host (Tavares et al. 2016;Jin, 2015;Li et al. 2018;Zidi et al. 2015;Lesiak et al. 2014). IL-10 is an antiinflammatory cytokine excreted by a variety of cells and has dual activity in the immune response by suppressing the cellular immune response (Th-1) and stimulating the humoral immune response (Th-2) (Mege et al. 2006). Patients with severe squamous intraepithelial lesions (SIL) have been found with increased levels of IL-10 expression (El-sherif et al. 2001;Zoodsma et al. 2005). TNF-α is a pro-inflammatory cytokine secreted mainly by macrophages, and has a key role in inflammation, immune homeostasis and host defense.
Furthermore, increased expression of adhesion molecules and activation of neutrophils stimulate the production of cytokines and act in a T-cell co-stimulatory activation and antibody production (Wajant, 2009). Additionally, TNF-α is involved in the defense against HPV infection by modulating viral replication (Zur Hausen, 2000).

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Polymorphisms in genes related with the immune response, such as IL-10 and TNF-α genes may be involved in the etiology of cervical disease (Tavares et al. 2016;Jin, 2015;Li et al. 2018;Zidi et al. 2015;Berrington et al, 2004). Several polymorphisms have been described in the IL-10 gene, in particular three in the promoter region (-1082 A/G, -819 T/C, and -592 A/C) may influence the transcriptional level of mRNA and protein expression in vitro and consequently they contribute to the development of cancer (Turner et al. 1997).
TNF-α also has several genic polymorphisms located in its promoter region: -1031 in the serum concentration of the protein encoded by this gene. The TNF-α -308 GG genotype was associated with low protein production, while -308 AA and -308 AG genotypes were associated with middle and high protein production, respectively (Fernandes et al. 2008).
In relation to cervical lesions, the presence of the TNF-α -308 GG genotype has been associated with the induction of squamous cervical intraepithelial lesions (SCIL), whereas the presence of TNF-α -308AG and TNF-α -308AA genotypes were associated with the progression to cervical lesions and even in the formation of invasive cervical cancer (ICC), however there have been conflicting results (Tavares et al. 2016;Jin, 2015;Crilly et al. 2003;Li et al. 2013).
Despite IL-10 -1082 (A/G) and TNF-α -308 G/A polymorphisms having been studied with respect to their susceptibility to many infectious diseases, the functional importance of these polymorphisms needs to be clarified. Therefore, this research aimed to investigate the possible association among IL-10 -1082 (rs1800896) and TNF-α -308 (rs1800629) gene polymorphisms and susceptibility to cervical intraepithelial lesions as well as to adenocarcinoma in a population from the Northeast of Brazil. and TNF-α -308 (G> A) genes were analyzed by the sequence-specific PCR technique (PCR-SSP). The analyses of the IL-10 -1082 (rs1800896) polymorphism gene were performed as described by Crilly et al. (2003), while analyses of the TNF-α -308 (rs1800629) polymorphism gene were performed as described by Perrey et al. (1999). Technologies) and 1 µM of each primer (common and specific allele). The cycling conditions used were the same as previously described in the literature cited above.

Statistical analysis
The genotype distribution and allele frequencies of the polymorphisms were obtained by direct counting. The Hardy-Weinberg equilibrium test was applied to datasets using the Biostat 5.0 (Manirauá, Amazônia, Brazil) program. Univariate statistical analyses and logistic regression were performed using the R software, version 3.0.2 (http://www.Rproject.org/). The Chi-square (χ2) test was used for analyzing categorical variables. The Student's t-test was used for continuous data with normal distribution and the Wilcoxon-Mann-Whitney test was performed for data without normal distribution. The Kolmogorov-Smirnov test was used to evaluate normality. The genotypes and their combinations were analyzed by the χ2 test and the odds ratio (OR) with 95 % confidence interval (CI). P-values under or equal to 0.05 were considered statistically significant.

Ethics
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
The local Ethics Committee for Research (nº 3326/13) approved this study.

RESULTS
In Table II are shown the distribution of allelic and genotypic frequencies of IL-10 -1082 A> G and TNF-α -308 G> A gene polymorphisms, between cases and healthy control groups. Individuals with the IL-10 -1082GG genotype and IL-10 -1082 G allele were significantly associated with increased risk for cervical lesions [OR = 2.17, p = 0.047; OR = 1.39, p = 0.037, respectively], as well as for the development of adenocarcinoma [OR = 3.8; p = 0.0029; OR = 2.19, p = 0.0002]. However, no significant difference in the distribution of allelic and genotypic frequencies were observed in relation to the TNF-α -308 G>A gene polymorphism, when compared with cervical lesions (p> 0.05) and adenocarcinoma (p> 0.05).
In Table III, we

DISCUSSION
In this study, we evaluated the correlation between the distribution of genotypic and allelic frequencies for both IL-10 (rs1800896) and TNF-α (rs1800629) polymorphisms and susceptibility to cervical lesions and development of adenocarcinoma. Our findings suggest that the presence of the GG and GA genotypic variants as well as the G allele in the promoter region of the IL-10 (rs1800896) gene influence the development of cervical lesions and adenocarcinoma. Some studies were conducted in order to verify the associations between IL-10 -1082 G/A polymorphism and cervical cancer and/or CIN, but the results have been inconsistent. Matsumoto et al. (2010) and Stanczuk et al. (2001) reported that, the IL-10 G allele was associated with a higher cervical cancer risk in Japanese and Zimbabwean populations, compared to IL-10 A allele. These findings can be explained by the increase in the transcriptional level of IL-10 and, as IL-10 is a cytokine produced by Th-2 cells and possesses immunosuppressive and antiangiogenic activities, it may lead to an increased susceptibility to both cervical lesions and adenocarcinoma. However, other studies did not find association of this allele with cervical cancer or CIN in Chinese (Wang et al. 2011;Yu et al. 2011), Korean (Roh et al. 2002, British (Farzaneh et al. 2006), Dutsh (Zoodsma et al. 2005) and Argentinian populations (Barbisan et al 2012). A possible explanation for different finds among the studies is heterogeneity of them (case definition and sampling, methods of genotyping, and differences in ethnicity).
In relation to the TNF-α -308 G/A gene polymorphism, it is known that the transcriptional level of mRNA of the TNF-α protein increases from 6 to 9 times in vitro in the presence of the transition from G to A (Cabrera et al 1995), and may affect the susceptibility to various diseases, including cervical cancer (Tavares et al. 2016;Pasha et al. 2013;Radwan et al. 2012;Gen-Selma et al. 2011;Kroeger et al. 1997). However, reports in the literature are still contradictory when related to susceptibility for cervical lesions and progression to cervical cancer. In this study, no association was found among the TNF-α -308 G/A gene polymorphism and the susceptibility to cervical lesions or development of adenocarcinoma (p> 0.05). Souza et al. (2014) studying a Portuguese population, also found no significant association with the development of pre-invasive cervical lesions. However, in this same study they found that individuals with the -308A allele and -308AA genotype had increased risk for developing cervical cancer. Kirkpatrick et al. (2004) found a significant association between individuals who were carriers of the GG genotype (low secretory) with the development of low-grade cervical intraepithelial lesions, but they did not find an association with respect to the development of cervical cancer. This last result agreed with others that have also shown no association (Wang et al. 2012;Govan et al. 2006). Furthermore, a meta-analysis performed by Liu et al. (2012) showed that individuals with African ancestry carrying the -308 AA genotype had been protected against developing cervical cancer, but no association in relation to Caucasian ancestry was observed. These discordant results may be explained by the difference in genetic background between the different populations analyzed.
Regarding socio-behavioral factors, our results showed that the IL-10 gene polymorphism was associated only with age above 35 years. On the other hand, when we analyzed the TNF-α -308 polymorphism, individuals carrying the GA and AA genotypes were significantly associated to several risk factors, such as age > 35 years, age at first intercourse <18 years, prolonged use of oral contraceptives (ACO), smoking, and presence of co-infection with CT (p< 0.05). Duarte et al. (2011) found similar results and suggested that these results could be explained by HPV latency in the body, causing histological changes in women of older age. Bezerra et al. (2005) showed that the early age of first sexual intercourse, considering the malformation of the female reproductive system contributes directly to an increase in the chance of developing cervical lesions. A study performed by Rosa et al. (2009) found that the use of ACO potentiates the onset of cervical lesions by about three times. In this same study, they found an increased risk for women with more parities.
Furthermore, Simonetti et al. (2009) suggested in their study that the co-infection by Chlamydia trachomatis may cause inflammatory responses that damage the cervical mucosa, leading to lesions or even facilitate the HPV infection. In our study, we found an association of IL-10 (rs1800896) polymorphisms with the predisposition to develop cervical lesions and adenocarcinoma. In addition, the clinical data of the patients presented significant differences for both IL-10 (rs1800896) and TNF-α (rs1800629) polymorphisms. Thus, our data suggest that the IL-10 and TNF-α genes can be used as molecular markers in patients predisposed to the screening of cervical disease.