Cytokines profiles in cervical mucosa in patients with cervical high-risk human papillomavirus infection

Introduction: The purpose of this study was to detect the expression of local cytokines in cervical mucosa between patients with transient and persistent HR-HPV infection with or without CIN. Methodology: A total of 150 patients who were diagnosed as HR-HPV infection in Tianjin Central Hospital of Obstetrics and Gynecology from January 2016 to December 2016 were included in this study. The expression levels of 9 cytokines in 150 patients with HR-HPV infection, including interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IL-12p70, IL-21, interferon (IFN)-γ and tumor necrosis factor (TNF)-α, were simultaneously measured by using a multiplex immunoassay. Moreover, HR-HPV genotype was performed by using pyrosequencing. The association between cytokines and HPV genotype was also investigated. Results: There was a statistically significant difference in IL-1β level between patients with HPV transient infection and HPV persistent infection (p = 0.041). There were statistically significant differences in the levels of IL-1β, IL-10, IL-21 and TNF-α between patients with low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) (p = 0.011, p = 0.008, p = 0.046 and p = 0.019, respectively). Conclusions: Pro-inflammatory cytokines, IL-1β and TNF-α, and Th2 type cytokines, IL-10 and IL-21, became stronger in cervical mucosa with the progression of CIN. IL-1β may be advantageous for HR-HPV persistent infection.


Introduction
Persistent high-risk human papillomavirus infection (HR-HPV) infection is an established causal factor for cervical intraepithelial neoplasia (CIN) and cervical cancer [1,2]. There is an annual incidence of 528,000 cases and mortality of 266,000 cases worldwide [3]. There are approximately 5% -10% women with HR-HPV infection who fail to clear the virus and develop to CIN [4]. Nearly 30% of low grade squamous intraepithelial lesion (LSIL) persists and 10% of LSIL progresses to high grade squamous intraepithelial lesion (HSIL). However, the specific mechanism of this phenomenon is unclear. It is postulated that HPV infection requires a tolerogenic local immune environment, involving avoidance or repression of both the innate and adaptive immune response [5]. The innate immune system contains epithelial barrier, antigen presenting cells (APCs), macrophages and natural killer (NK) cells. Generally speaking, APCs process pathogens and provide pathogen related molecular patterns (PAMPs) to the pattern recognition receptors (PRR) located on immune cells. It has been reported that PAMPs can combine with PRRs to activate a cascade of NF-κB pathway, which has the ability to enter nucleus and induces the expression of different genes. Those genes include cytokines, chemokines and others that involve in defensing pathogenic invasion [6,7]. However, the function of APCs to recognize L1 protein of HR-HPV is blocked by HPV-infected keratinocytes, which contributes to HPV escape from host immunity and persistent infection.
The dose of response mode is an important part of adaptive immune system. It includes cell-mediated type 1 reactions, characterized by interleukin (IL) -12 produced by macrophages and dendritic cells, interferon (IFN) -γ produced by NK cells and activated T cells, and proinflammatory cytokines such as IL-6, IL-8, macrophage inflammatory protein (MIP) -1α and tumor necrosis factor (TNF). Oncoprotein of HPV inhibits IFN secretion and promotes production of transforming growth factor (TGF)-β and IL-10, which belongs to cytokines produced by type-2 response and acts as immunosuppressive cytokines [8]. HPV has established immune tolerance and special immune microenvironment for the infection and progression of CIN.
Therefore, the purpose of this study was to evaluate the relationship between cytokines produced locally in the cervix and the status of HR-HPV infection and the levels of cervical precancerous lesion. Then cytokines which were significantly different were screened out in patients with transient and persistent HR-HPV infection and with different levels of cervical lesion. Moreover, the mechanisms of HR-HPV persistent infection and progression of cervical neoplasia were analyzed to provide immunity theory for blocking HR-HPV persistent infection and progression of cervical lesion.

Study population
This study was approval by the Ethics Committee of Tianjin Central Obstetrics and Gynecology Hospital. From January 2016 to December 2016, 150 patients who were diagnosed as HR-HPV infection by Hybrid Capture II (HCII) in Tianjin Central Obstetrics and Gynecology Hospital were enrolled in this crosssectional study. Exclusion criteria: (1) pregnant or postpartum within the previous 6 months; (2) patients who had history of hysterectomy or invasive cervical procedure; (3) patients who had cancer chemotherapy; (4) patients who had sexual behavior within 3 days; (5) chronic diseases, such as diabetes mellitus, allergy, autoimmune diseases; (6) patients who had vaginal and cervical medication within 1 week; (7) patients who had oral contraceptives and other cervical pathogens infection. All patients were officially informed and gave their written consent. Data on epidemiology were collected by survey.
A total of 36 patients who had twice consecutive negative HR-HPV test after at least one positive HR-HPV infection were defined as transient HR-HPV infection. A total of 40 patients who had more than three times consecutive positive HR-HPV infection were defined as persistent HR-HPV infection. Both patients with transient and persistent HR-HPV infection in this study were diagnosed as having no cervical lesion by biopsy. The other patients who had both HR-HPV infection and cervical lesion were divided into 2 subgroups: LSIL and HSIL groups, including 36 and 38 patients, respectively.

Sample collection
Two kinds of sample were selected in our study, including cervicovaginal lavage (CVL) and cervical exfoliated cells. They were used for testing cytokine and HR-HPV type, respectively. CVL specimens were obtained by washing the cervix with a single 5 mL volume of normal saline and were collected before all other cervical samples. The collected CVL aliquots were centrifuged (3000 rpm) for 10 minutes to separate mucus and cellular components. Cell-free supernatant was separated into a 1.5 mL aliquot and frozen at -80 °C until use. The cervical cells were collected by brush of liquid-based cytology and transferred directly into a special Thin-Prep container (ThinPrep Pap test; Hologic), which were stored at 4 °C until detection.

Assay and Typing of HR-HPV
Thin-Prep samples containing cervical cells were used for DNA extraction using the TIANamp Micro DNA kit (TIANGEN Biotech Co. Ltd., Beijing). Extracted DNA samples were tested for HPV type by polymerase chain reaction (PCR) assay and targeting the L1 region of the viral genome by using PGMY primers [11]. Amplification of human β-globin gene segment was used as an internal control for DNA quality and cases negative for β-globin were excluded. Then the final PCR production were genotyped using pyrosequencing which was described previously [12]. Each sample was evaluated for the presence of high-risk HR-HPV types (16,18,31,33,35,39,45, 51, 52, 56, 58, 59, 66, 68 and 69). Transient infection samples of positive HPV typing and persistent infection samples of negative HPV typing did not carry on follow-up analysis.

Cytokine Measurement
The levels of 9 cytokines (IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12p70, IL-21, IFN-γ, and TNF-α) were simultaneously measured by using multiplex immunoassay and tested using MilliPlex MAP Human Cytokine/Chemokine immunoassay kits (Millipore corporation, Billerica, MA). A standard curve was made according to the manufacturer's instructions. Briefly, samples (25 μL per well) were incubated with antibody-conjugated microspheres in 96-well filtermembrane assay plates with agitation overnight. Plates were then washed with buffer provided in the assay kits and vacuum filtration. Besides, analyte-bound beads were incubated with a biotinylated detection antibody cocktail and finally with streptavidin-phycoerythrin. After additional wash steps and resuspension of beads in instrument sheath fluid, plates were run on Luminex 100 instrument (Luminex, Austin, TX). Regression curves (5-parameter logistic) were fit, and unknown concentrations were determined in pg/mL by using MiraiBio MasterPlex QT version 2.5 analysis software (Hitachi Solutions America, Ltd., South San Francisco, CA).

Statistical Analysis
Statistical analysis was performed using software SPSS 22.0 (International Business Machines, corp., Armonk, NY, USA). The independent sample t test (parametric) was used for analysis if there were differences in the mean value of 9 cytokines between two groups. p < 0.05 was considered to be significantly different in all analyses.

General information of patients
The average age of the 150 patients was (48.81 ± 3.67) years. There were 31 menopausal women in this study. There was no significant difference in age among the four groups (p > 0.05). All patients had HR-HPV (HC-2) test report and cervical biopsy report.
Furthermore, the levels of each cytokine in each group were shown in Table 2. Due to the low relevance ratio (< 60%), IL-2, IL-4, IL-12 and IFN-γ were excluded in analysis. There were significant differences in the levels of IL-1β between transient HR-HPV infection group and persistent HR-HPV infection group (p = 0.041) (Figure 1). Moreover, there were obvious differences in the levels of IL-1β, IL-10, IL-21 and TNF-α between LSIL group and HSIL group (p = 0.011, p = 0.008, p = 0.046 and p = 0.019, respectively) ( Figure 1).
The mean values of cytokines of three categories in transient HR-HPV infection group, persistent HR-HPV infection group, LSIL group and HSIL group were shown in Figure 2. There was notable difference in the levels of cytokines of various category in the four groups (p > 0.05). The levels of pro-inflammatory cytokines were significantly increased in comparison those of regulatory factors of lymphocytes and regulatory factors of immune inflammation in the four groups, respectively (p > 0.05). However, there were not statistically significant differences between regulatory factors of lymphocytes and regulatory factors of immune inflammation in the four groups (p < 0.05, respectively). Similarly, there were not statistically significant differences in pro-inflammatory cytokines, regulatory factors of lymphocytes and regulatory factors of immune inflammation among the four groups (p < 0.05, respectively).

The prevalence of 15 types HR-HPV infection
All specimens of patients with persistent HR-HPV infection and patients with transient HR-HPV infection were positive. A total of 10 out of 15 HR-HPV genotypes were detected in our study (Table 3). These 10 kinds of HPV were included in the detection range of HCII. In 114 patients with persistent HR-HPV infection, 94 infected with one type of HR-HPV, 16 infected with two types of HR-HPV and 4 infected with three types of HR-HPV.  There were no significant differences in the levels of various cytokines in different HR-HPV types (the data was not shown). Similarly, no significant differences were found in the levels of cytokines between HR-HPV-16 and other HR-HPV types (the data was not shown).

Discussion
In this cross-sectional study of women with HR-HPV infection, we analyzed the levels of 9 cytokines in cervical mucosa between patients with transient HR-HPV infection and persistent HR-HPV infection, and between patients with LSIL and HSIL. In this study, increased levels of IL-8, IL-1β, IL-10, IL-21 and TNFα were associated with the progression of cervical precancerous lesions. It suggested that various types of immune cells secreted multiple cytokines to participate in cervical HR-HPV infection and progression of cervical lesion broadly.
HR-HPV infection is an essential factor in inducing cervical lesion. Once HPV infected cervical epithelial cells, the immune response of host involved in the removal of HPV. The monocytes and macrophages, which constitute the host innate immunity, are activated and result in raised levels of IL-1β that favor to maintain HPV infection [9]. With the assistance of proinflammatory cytokine, HPV may occur innate immune escape.
In cervical precancerous lesion with HR-HPV infection, the number of macrophages are increased, CD4 + and CD8 + T cells gather in the cervix and secrete magnanimous pleiotropic cytokines, such as IL-1β, IL-10, IL-21 and TNF-α. IL-1β and TNF-α are typical representative of pro-inflammatory cytokines [13]. IL-10 and IL-21 belong to Th2 type cytokines, and the low detection of Th1 type cytokines may imply the correlation of shift of Th1/Th2 and lesion progression.
Many studies [9,14,15] showed that high levels of IL-1β and TNF-α related to the occurrence and development of cervical lesions. Both normal cervical and lesion cells can produce IL-1β and TNF-α [16], and they in turn stimulate lesion cell proliferation and even cancelation. With the binding of IL-1β and TNF-α with their respective receptors, p38 MAPK and NF-κB signaling pathway can be activated. Followed by production of chemokines, vascular growth factor and tumor growth factor provide favorable immune microenvironment for the survival of tumor cells and progression of lesion. Additionally, the circulating levels of TNF-α were increased with the increased clinical stage progress, and expression levels of KI-67, Bcl-2 and p53 in stage Iβ-IIa of cervical carcinoma were positively correlated with the levels of TNF-α [17].
As a potent immunosuppressive factor, IL-10 plays an extremely essential function in the course of HR-HPV infection and cervical precancerous condition. High expression of IL-10 in HPV persist infection may be beneficial to the immunoescape of HPV [15]. And, it can promote the occurrence and development of tumor cells by activating STA3 pathway and play the role of immune suppression [16], which can explain our results of IL-10. Besides, in response to (lipopolysaccharide) LPS, the levels of IL-10 were upregulated accompanied with the decreased levels of IL-12 [18], which inhibited the anti-proliferative effect of IL-12 [19].
In addition to IL-10, IL-21 is also a kind of Th2 type cytokine. IL-21 is a pleiotropy cytokine, which be secreted by NKT cells, CD4 + and CD8 + T cells, and IL-21 in turn enhances the cytotoxicity of them. It can activate conventional dendritic cells and then induces IL-1β expression. In addition, it can promote M2 to M1 transition of the tumor-associated macrophages, followed by increased levels of IL-10 and chemokines [20]. In view of its wide range of influence on immune cells and cytokines, it may play a central role in immune response. At present, there are few studies on IL-21 in cervical area. Research showed that combination of IL-21 and IL-2 could availably stimulate peripheral blood mononuclear cells with cytotoxicity against SiHa cells, which may be the mechanism owing to down-regulated Treg and Th17 cell differentiation [21].
Contrast with regulatory factors of lymphocytes and regulatory factors of immune inflammation, the expression of pro-inflammatory cytokines was higher in the four groups. Pro-inflammatory cytokines may occupy an important position in cervical immune micro-environment [16], and have profound effect on persistent HR-HPV infection and cervical lesion. Certain study showed that pro-inflammatory cytokines were highly expressed in persistent HPV infection, Table 3. The incidence of HR-HPV types in the patients with LSIL and HSIL but without cervical lesion. HR-HPV type  16  18  31  33  39  51  52  56  58  68  No cervical lesion  9  14  3  2  2  1  7  3  3  2  LSIL  13  10  3  1  1  -4  2  6  1  HSIL  23  12  -1  --1  5 8 -which then caused the negative regulation of p53 and promoted proliferation of tumor cells and formation of blood-vessels [22]. Although there was no statistical difference in the levels of pro-inflammatory cytokines in the four groups, their roles cannot be ignored. As a cross-sectional study, there are some limitations in our study. Firstly, participants in our research had HR-HPV infection before, but we failed to get the samples of which microenvironment can regard as base-line level and dynamic variation process. It may reduce convincing of the results. Secondly, the essence of our samples for cytokines detection, CVLs, was dilution, which may limit measurement of low-level cytokines. Diverse approaches have been employed in studies of cervical immune markers, including ophthalmic sponges, aspirates and CVLs [15]. Moreover, the reason why we used CVLs for detection of immunoglobulin in this study was that we did it in our previous study. Contrasted with ophthalmic sponges, CVLs indeed processed a low detection rate and concentration [23]. Lastly, there were no explicit definitions of cervical HR-HPV transient and persistent infection at present.
Nowadays, there are multiple means of test on cytokines and HPV typing. On the term of cytokines, both mRNA level of cervical tissue cytokine and peripheral blood mononuclear cells of patients with cervical HPV infection could be used for test cytokines. Whereas, the mRNA level may not reflect the protein level and systemic immune response does not represent. Therefore, detection of cervical local protein levels is appropriate for local immunity cytokine test. On the term of HPV typing, new technology emerges endlessly, such as HCII and Combas 100 based on DNA detection, Aptima targeting HPV mRNA from the E6/E7 oncogenes, and so on. Pyrosequencing, a new technology targeting HPV L1 capsid protein, processes equal sensibility and specificity compared with HCII.
In conclusion, the increased local levels of proinflammatory cytokine (IL-1β) was associated with the decreased possibility of clearance in our study. The levels of IL-8, IL-1β, IL-10, IL-21 and TNF-α were found to be lower in patients with LSIL than those in patients with HSIL. Cytokines may reflect cervical mucosa immune response against HR-HPV, and they can be used as biological markers for the HR-HPV infection and prognosis of CIN. However, further researches with follow-up visits and large numbers of subjects were requisite for exact mechanism.