The existence of Th22, pure Th17 and Th1 cells in CIN and Cervical Cancer along with their frequency variation in different stages of cervical cancer

Background Recently, it is found that T-helper (Th) 22 cells are involved in different types of autoimmune and tumor diseases. But, till now, no study has been carried out to understand the involvement of these cells in cervical cancer (CC). Methods Flow cytometry was used to determine the expression of interferon gamma (IFN-γ), Interleukin-22 (IL-22), IL-17 in the peripheral blood of healthy controls (HC), CIN and cervical cancer patients. From peripheral blood mononuclear cells (PBMCs), mRNA expression levels of Aryl hydrocarbon receptor (AHR), RAR-related orphan receptor C (RORC), TNF-α and IL-6 were respectively determined. Using the method of ELISA, plasma concentrations of IL-22, IL-17 and TNF-α were examined. Results Th22 and Th17 cells were elevated in CC and CIN patients. Th1 cells and the plasma concentrations of IL-22 in CC patients were significantly increased compared with HC. In CC patients, an increased prevalence of Th22 cells was associated with lymph node metastases. There was a positive correlation between Th22 and Th17 cells, but an approximately negative correlation between Th22 and Th1 cells in CC patients. The mRNA expression of RORC, TNF-α and IL-6 was significantly high in CC patients. Conclusions Our results indicate that there is a higher circulatory frequency of Th22, Th17 and Th1 cells in CC which may conjointly participate in the pathogenesis and growth of CC.


Background
Cervical cancer (CC) is one of the leading gynecological cancers in developing countries. The main etiology behind this occurrence is the persistent infection of high-risk human papillomavirus (HPV) [1][2][3]. Even if the incidence of HPV is high, with the help of cell mediated immunity, it can be cleared spontaneously [4][5][6]. A very few cases may develop into advanced CC from precancerous lesions which may indicate a substantial role of immune regulation in the controlling of HPV associated lesions and cancer progression [7].
We know that T helper (Th) cells, one subgroup of lymphocytes, have an essential role in the immune system. Recently it was demonstrated that Th cells such as Th1, Th2, Th17, Treg cells, participate in the pathogenesis and progression of different solid tumors [7][8][9][10]. A newly discovered T cell subset -Th22 cells, which were detected in autoimmune and inflammatory diseases, have the ability to secrete IL-22 and TNF-α, but do not express IL-4 (Th2 marker), IL-17 (Th17 marker) or IFN-γ (Th1 marker). In the human body (in the presence of IL-6 or/and TNF-α) the naive CD4 + cells differentiate into Th22 cells with the aid of plasmacytoid dendritic cells, AHR and RORC [11,12]. It is known that Th22 is a distinct subset with novel characteristics compared to other Th cells (Th17, Th2 and Th1 cells). It is demonstrated that Th22 cells play an important role in the pathogenesis of inflammatory diseases and autoimmunity diseases such as psoriasis, Graves' disease and rheumatoid arthritis. [13][14][15]. However, the nature of Th22 cells are not properly umderstood in human cancer. Recently some studies concluded that Th22 cells contribute to the the progression of hepatocellular and gastric carcinoma which indicates that Th22 cells may be involved in the development of tumors. [16][17][18].
IL-22 which belongs to the IL-10 cytokine family is mainly an effector cytokine of Th22 cells. IL-22 maintains its function by binding to a heterodimeric transmembrane receptor complex consisting of IL-10R2 and IL-22R1, and activates Janus kinase (signal transducers and activators of transcription signaling pathways which acts with a dual role in inflammatory and autoimmune diseases [19][20][21]). It is seen that IL-22 leads to tumor proliferation, apoptosis suppression and metastasis promotion by activation of STAT3 in colon cancer [22]. Reversely, IL-22 exerts a protective role in mucosal wound healing acceleration by inducing STAT3-dependent expression in ulcerative colitis [23,24].
To the best of our knowledge, no previous study has shown data that considers Th22 cells and their association with Th17 or Th1 in cervical cancer. To examine the possible status of these cells in the pathophysiology of CC, we measured the frequency of peripheral Th22, Th17, Th1, mRNA expression levels of RORC, AHR, IL-6, TNF-α in PBMCs along with plasma concentrations of IL-22, IL-17 and TNF-α in PB of CC, CIN patients and HC for assessing their relevance.

Patients and controls
Six-one pathologically confirmed CC patients (age 24 -60 years, median 48 years) and 38 CIN patients (age 27-61 years, median 42 years) were enrolled in this study. All the patients of CIN group have biopsy results of CINIII. Before the study none of the patients had received anticancer treatment.
Thirty-two healthy women with normal results of pap smear (TCT) and HPV (HC2) tests served as controls (age 22-47 years, median 27 years).They are from our Gynecologic Clinic and Regular Physical Examination Center.
The participants with simultaneous active or chronic infection, autoimmune disease, diabetes, or a history of other malignant tumors or connective tissue diseases were excluded. The characteristics of the patients are given in   100 μl of Reagent B (FIX &PERM Kit, MultiSciences Biotech Co., Ltd.) and the recommended dose of anti-IL17A-PE and anti-IL22-APC and anti-IFNγ-FITC monoclonal antibody were added to each sample after fixation and permeabilization. Samples were then incubated at RT in the dark for 15 min to stain IL-17, IL-22 and IFN-γ. Isotype controls were used to correct compensation and confirm antibody specificity. After washing the cells with 3 ml PBS, we added 300 μl PBS to re-suspend the cells for cytometric analysis. Stained cells were analyzed by flow cytometric analysis using a FACS cytometer equipped with Cell Quest software (BD Bioscience Pharmingen). All antibodies mentioned above were from eBioscience (San Diego, CA, USA). Th17, Th22 and Th1 cells are defined as CD4 + IFNγˉIL17 + IL22ˉ, CD4 + IFNγˉI-L17ˉIL22 + and CD4 + IFNγ + cells respectively.

Quantitative real-time PCR analysis
Trizol (Invitrogen, America) was used for isolation of Total RNA from PBMCs. For reverse transcription reaction the Prime Script RT reagent kit (Perfect Real Time; Takara) was used according to the instruction of the manufacturer. Reverse transcription reaction was done at 37°C for 15 minutes, followed by 85°C for 5 seconds.

Statistical analysis
Mean ± SD or median (range) were used for expression of values. Distribution of the data was obtained from Kolmogorov-Smirnov test (K-S test). ANOVA and Newman-Kuels multiple comparison tests were used for the assessment of normal distribution data. Kruskal-Wallis test (H test) and Nemenyi tests were used for unusual data. Assessment of Correlation analysis was obtained from Pearson correlation. A p value less than 0.5 was considered statistically significant. All tests were performed by SPSS 17.0 software. . Besides, significant difference was also found in Th22 cells between CIN and CC patients (p < 0.001), but none in Th17 cells ( Fig. 2a and b).
A typical dot plot of the percentage of Th1 cells, Th22 cells and Th17 cells in representative patients and HC is shown in Fig. 1.

Discussion
Persistent infection with HPV is the main cause of CC and CIN [25,26]. That CIN and CC arise more frequently in immunosuppressive women indicates that elimination of HPV is related to immunity function. In the evolution of these diseases, local or systemic immune mechanisms abnormalities may be involved [27,28]. A vast and dynamic crosstalk among immune cells, along with cytokines turmoil has been regarded as a crucial element of cancer pathophysiology [29]. In our current study, we focused on immune cells, mainly three subtypes of T helper cells-Th1, Th17 and Th22 cells and their probable role in CC and CIN.
Interferon (IFN)-γ causes activation of immune cells in the tumor microenvironment. It is known that Th1 cells, the main source of IFN-γ, have a powerful anti-tumor function. To enhance the function of antigen presenting cells, tumor antigen specific CD4 + Th1 cells can travel to the tumor site and secrete inflammatory cytokines and modulate the microenvironment [30]. It was observed that for cancer inhibition and better outcomes, Th1 adaptive immunity is essential [31]. In our study, we demonstrated a significant elevated frequency of Th1 cells in CC patients, compared to CIN patients and HC, which is consistent with other previous studies of the involvement of Th1 cells in tumors.
It was noticed that another two inflammatory cell subgroups, Th17 and Th22 cells are involved in viral infection and mucosal immunity [32,33,34]. In our previous study, we saw that there was a significant increase of Th17 cells (CD4 + IL17 + cells) in CIN and CC patients [8,11]. In order to exclude multiple positive cells Th17 cells are defined as CD4 + IFNγ − IL17 + IL22 − cells, which also were called as "pure Th17 cells". Th22 cells are now defined as CD4 + IL17ˉIL22 + IFNγ − cells, which is an independent subset of T helper cells from Th1 and Th17 cells [35][36][37]. In the current study, we evaluate the frequencies of pure Th17 and Th22 cells to confirm the probable role of these two famous types of T helper cells in PB of CIN and CC by flow cytometry. As expected, increased frequencies of Th17 and Th22 cells were found in both CIN and CC compared to HC. Moreover, the increased change of Th22 cells in CC was much higher than that of CIN. It suggested that as cervical precancerous lesion occurs, Th22 cells might gradually elevate from CIN to CC. However, no significant difference of Th17 cells was found between CIN and CC. But the data indeed shows that there are frequencies of Th17 and Th22 cells changed in the tumorigenesis of both CIN and CC which indicate these two types of cells may paticipate in tumor immunity.
IL-22 is known to have a relationship with virusinfection reactions and whose receptor is confined to nonhematopoietic cells (mainly epithelial cells). Previously it was considered that IL-22 is a cytokine of Th17 cells. Now it is considered as the characteristic product of Th22 cells. Our study also revealed elevated levels of plasma IL-22 in CC patients. Additionally, expression of a series of molecules, which are responsible for cellular differentiation and survival was triggered by IL-22 [38,39]. In our study, a raised level of plasma IL-22 was found, which indicated that Th22 cells, the main T helper cells which product IL-22, may be involved in the process of CC. However, plasma IL-17 did not show a significant change. This might be due to the fact that concentration of IL-17 was too low to present the change, as it showed low levels in both of CC and HC. In addition, there was a positive correlation between the frequencies of Th17 and Th22 cells in CC patients, suggesting that differentiation of Th22 cells may be linked to Th17 cells or even Th22 cells might partly derive from Th17 cells. This derivation may partly explain the type of IL22 + Th17 cells. However, no correlation was found in CIN III or HC. One reason for this is that the frequencies level of Th17 and Th22 cells are very low, hence the difference between detected results and real conditions multiplied and distorted the statistic results. Another reason is that, in a normal situation, Th17 and Th22 cells are derived from a different origin and induced by different stimuli. However, when cancer appears, inflammatory cells show a partly inter-related differentiation, which also causes elevated frequency of IL22 + Th17 cells during the process.
It was seen that RORC is the key transcription factor directing Th17 lineage and modulates the polarization of Th22 cells [12,40]. In our study we noticed a notably elevated expression of RORC in CIN and CC patients.  Fig. 6 The Th22, Th17 or Th1 cells frequency in positive or negative lymph node metastases. Increased frequency (p = 0.026) of Th22 was observed in CC patients with lymph node metastases (2.20 ± 0.85 %, n = 11) comparing to CC patients without lymph node metastases (1.68 ± 0.64 %, n = 48). *p < 0.05, NS no significance Also, the expression of RORC is positively correlated with both Th22 and Th17 cells. It is assumed that in CIN and CC patients the differentiation of Th22 and Th17 cells is mainly regulated by RORC. We previously found that IL-6, which promoted differentiation of Th22 cells, is highly expressed in CIN and CC patients [11,12,19]. Elevated IL-6 mRNA expression was found in CIN and CC patients compared to HC. The data showed that, in CC and CIN patients, immune environment may be more suitable for polarization of Th22 cells.
However, no significance was found in AHR expression. Although AHR is the most important transcription factor of Th22 cells, AHR pathway is not unique. It is demonstrated that TGF-β could inhibit IL-22 secreting of Th17 cells by AHR-independent pathways. In our study of CIN and CC, no significant change was found. The explanation for increase of Th22 cells may not be caused by AHR (transcription level), but others pathways, such like stimulation and transformation.
Referring to clinic factors, in CC patients, lymph node metastases were found to correlate with aggregation of Th22 cells. Again, a positive association between Th22 cells and Th17 cells was also observed. Consequently, it is imaginable that co-increased levels of Th22 and Th17 cells along with pro-inflammatory cytokines may play a synergistic role in the progression of CC. Nevertheless, there was an approximately negative correlation between Th1 cells and Th22 cells in CC patients. This argues that the beneficial Th1 cells gradually declined while more Th22 was produced toward disease progression. However, the interaction among these three different cells demands further investigation.

Conclusion
It is seen that patients with CC possess a high frequency of circulating Th22 cells, Th17 cells and Th1 cells. The higher prevalence of Th22 cells was found in patients with advanced CC, arguing an important role for this T-cell subtype in the growth and acceleration of CC. For a better understanding of this development (i.e., regulation and function of these cells in CC) more extensive experiments are needed which may lead to the evolution of promising therapeutic strategy for CC patients.