IL‐6 expression promoted by Poly(I:C) in cervical cancer cells regulates cytokine expression and recruitment of macrophages

Abstract Poly(I:C) is a promising adjuvant for cancer treatment vaccines to enhance the host anti‐tumour immune response. However, the roles of poly(I:C) in the cervical cancer microenvironment and local immune reactions are not well understood. In this study, we investigated the roles of poly(I:C) in the cervical cancer. We analysed the cytokine transcription and secretion of cervical cancer cell lines and THP‐1–derived macrophages after poly(I:C) treatment, respectively. These results revealed that IL‐6 was significantly up‐regulated, and this up‐regulation was partly dose dependent. poly(I:C)‐stimulated supernatant of cervical cancer cells promoted M1‐type cytokine IL‐1β and IL‐6 expression of THP‐1–derived macrophages, but inhibited the expression of M2‐type cytokine, IL‐10 and CCL22. The recruitment of THP‐1–derived macrophages by poly(I:C)‐stimulated cervical cancer cell supernatant was also enhanced. Inhibition of IL‐6 expression in cervical cancer cells by siRNA transfection almost completely reversed the effects of poly(I:C) treatment. Finally, we found that phosphorylation of the NF‐κB signalling pathway in cervical cancer cells occurred quickly after poly(I:C) treatment. Moreover, the NF‐κB signalling pathway inhibitor PDTC significantly inhibited poly(I:C)‐induced IL‐6 expression. Taken together, these results suggest that poly(I:C) might regulate the effects of cervical cancer cells on tumour‐infiltrated macrophages, and subsequently promote a pro‐inflammatory tumour microenvironment.

acid (poly(I:C)), a synthetic compound that mimics viral dsRNA polymers, is one of the most extensively studied adjuvants of anti-tumour vaccines. 2 As the ligand of the Toll-like receptor 3 (TLR3), poly(I:C) triggers the procession and presentation of antigens in innate immune cells, including macrophages and dendritic cells, subsequently leading to anti-tumour adaptive immunity. 3,4 Elevated immunological responses have been observed in various poly(I:C)-involved strategies over the past decade. In human papillomavirus (HPV)-infected genital tumours, the application of poly(I:C), along with HPV16E7 peptide vaccination, has increased, by approximately fivefold, the number of vaccine-specific CD8+ T cells infiltrated in the genital mucosa of mice. 5 This observation is particularly interesting, considering that the levels of inflammatory cytokine expression and immunocyte infiltration were usually low in high-risk HPV-infected lesions, especially in cervical cancer, due to the immunosuppression effect of HPV oncoproteins. [6][7][8] One possible explanation was that poly(I:C) modulated the tumour microenvironment, an established mechanism lacking detailed investigation.
The tumour microenvironment contains various myeloid lineage cells that usually show unique tumour-supportive characteristics compared with their companion residents in normal tissue. In cancer vaccination, tumour-derived nucleic acids, such as poly(I:C), act on both tumour cells and non-malignant components, resulting in a complex interaction network. Previous research has shown that the cytosolic effect of poly(I:C) evokes an inflammatory form of death in tumour cells, as well as several other effects including type I IFN induction and myeloid cell maturation and activation. 9 In normal keratinocytes, which are host cells of HPV, extracellular poly(I:C) stimulation greatly induced inflammatory mediator expression, including tumour necrosis factor α and type I IFNs, therefore promoting the activation of dendritic cells. 10 Similar observations were also achieved in cervical cancer cells in which poly(I:C) enhanced the expression of necroptosis regulator RIPK3, and increased IL-12 production in dendritic cells. 11 However, the effects of poly(I:C) on the interactions between the tumour microenvironment and tumour-infiltrated macrophages, the number of which was almost ten times higher than that of dendritic cells, 12 have not been fully explored.
In this study, we demonstrate that cervical cancer cells stimulated by poly(I:C) regulate cytokine expression and recruitment of THP-1-derived macrophages. The enhanced IL-6 expression in cervical cancer cells through the NF-κB signalling pathway was shown as a critical factor in the modulation of poly(I:C)-stimulated cervical cancer cells on macrophages.

| Cell culture and generation of THP-1-derived macrophages
Human cervical cancer cell lines HeLa and CaSki and acute monocytic leukaemia cell line THP-1 were obtained from the American Type Culture Collection (ATCC). The cells were cultured in RPMI-1640 medium supplemented with heat-incubated 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in an incubator with 5% CO 2 .
To obtain the conditioned medium (CM) of HeLa and CaSki cells, 2 × 10 5 HeLa and CaSki cells were seeded into 2 mL RPMI-1640 medium/10% FBS into a 6-well plate. After 6 hours, different concentrations of poly(I:C) were added to the medium. The medium was then washed and replaced with new RPMI-1640 medium with 10% FBS for another 12 hours. Cell supernatant was collected as HeLa and CaSki CM for later experiments.
THP-1-derived macrophages were generated as previously described. 13 Precisely, 1 × 10 6 THP-1 cells were treated with 100 ng/ mL PMA for 48 hours and adherent cells were collected for later experiments. In some cases, the medium was replaced with 50% total volume of the CM of cervical cancer cells after 6 hours of treatment with phorbol myristate acetate (PMA), and THP-1 cells were further cultured for another 42 hours with the concentration of PMA maintained at 100 ng/mL.

| Real-time quantitative RT-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen), and the cDNA was synthesized by reverse transcription. Quantitative RT-PCR was performed on a LightCycler 2.0 (Roche Diagnostic). GAPDH was used as an internal control. The mRNA level of each sample was measured by the 2−ΔΔT method. The primer sequences were listed in the supplementary documents (Table S1).

| Small interfering RNA transfection
Small interfering RNA (siRNA) targeting human IL-6 and a scramble-negative control was designed by GeneChem Co., Ltd.    (12 h) was tested by ELISA. The mRNA or protein level of each cytokine in poly(I:C)-treated group was compared with the corresponding control group. Each bar represents mean ± SD (n = 3. *P < .05; **P < .01. ⋆, below minimum detection limit)

| Statistical analysis
All experiments were performed three times. The statistical analyses were performed, and experimental graphs were generated using SPSS 17.0 and GraphPad Prism software, respectively. Descriptive statistics, including the mean ± SD and paired/unpaired Student's t test and one-way ANOVA tests, were used to analyse the significance of differences. P values of <.05 were considered significant (*P < .05, **P < .01). We further explored whether the regulation of poly(I:C) on IL-6 expression in cervical cancer is related to its concentration and duration of action. As shown in Figure 2A

| Poly(I:C) regulates the effect of cervical cancer cells on the secretion of cytokines from THP-1derived macrophages
To investigate the effect of poly(I:C)-treated cervical cancer cells on macrophage cytokine expression profiles, we collected conditioned medium (CM) with or without poly(I:C) treatment. The CM was added during the inducing differentiation of the THP-1 monocyte line into macrophages by PMA. The treatment procedure was described in the Methods section above. As shown in  We further explored the role of IL-6 in the promotion of macrophage recruitment by cervical cancer cells in poly(I:C). (Figure 5D,E).

| Poly(I:C) promotes recruitment of THP-1derived macrophages by cervical cancer cells
The recruitment of HeLa cell CM transfected with IL-6 siRNA to THP-1-derived macrophages was significantly inhibited compared with control siRNA.

| Poly(I:C) promotes the expression of IL-6 in cervical cancer cells via the NF-κB pathway
NF-κB is a signalling pathway closely related to inflammatory reactions. In this study, we found that the level of phosphorylated NF-κB in HeLa cells was up-regulated 5 minutes after poly(I:C) treatment, reaching a peak at 30 minutes, suggesting that the signalling pathway is activated ( Figure 6A). To investigate whether the activated NF-κB signalling pathway is involved in poly(I:C)-regulated IL-6 expression, we selected the common NF-κB signalling pathway inhibitor PDTC (4 μg/mL) to pre-treat HeLa cells for 1 hour, then changed the medium and added poly(I:C). ELISA results showed that PDTC significantly reversed the effect of poly(I:C) on IL-6 secretion in HeLa cells ( Figure 6B).  recruitment of tumour-promoting Th17 cells to the tumour site. 28 The above results suggest that IL-6 is one of the key molecules connecting local chronic inflammatory response and tumour development. 29 Our results show that poly(I:C) up-regulated cervical and TNF-α. 32 Another study showed that the severity of insulitis in diabetes-prone BioBreeding (BB) rats caused by poly(I:C) was dose-dependent; however, low-dose poly(I:C) (0.05 mg/g body weight) was shown to be effective in preventing the transcription of TNF-α in splenocytes and the development of insulitis. 33 In this study, we noted that there is a correlation between the expression level of IL-6 in cervical cancer cells and poly(I:C) concentration.

| D ISCUSS I ON
When poly(I:C) concentration was 1 μg/mL, IL-6 expression was significantly promoted, but the secretion level was significantly lower than that of the 25 μg/mL poly(I:C) treatment group. This suggests that the dose of poly(I:C) should be carefully selected before application in tumour treatment to maintain its immune activation characteristics while avoiding its potential risk of promoting tumour development.
In conclusion, we found that poly(I:C) promoted the secretion of IL-6 by cervical cancer cells through activation of the NF-κB pathway. The elevated IL-6 level in cervical cancer supernatant regulated the secretion profile of THP-1-derived macrophages and promoted the recruitment of macrophages by cervical cancer supernatant.
This finding suggests that poly(I:C) as an adjuvant may enhance the local inflammatory response in cervical cancer, but also could be a F I G U R E 6 Poly(I:C)-promoted IL-6 expression was depend on activation of NF-κB signalling pathway. (A) Phosphorylation of NF-κB (pNF-κB) and total NF-κB was measured at different time-points after poly(I:C) stimulation (upper), and the ratio of pNF-κB and total NF-κB was calculated (lower). GAPDH was analysed as loading control. (B). The IL-6 concentration of HeLa cells pre-treated by NF-κB inhibitor, PDTC, with/without poly(I:C) stimulation. Each bar represents mean ± SD (n = 3. **P < .01.) risk in cervical cancer immunotherapy. Measures such as appropriate application doses should be taken into consideration to control its effects on non-immune cells and reduce the risk of tumour development. Despite this, there is still much to be determined in our research. For example, does poly(I:C) affect the anti-tumour activity of local-infiltrated macrophages by IL-6? Does poly(I:C) have a similar affect in other types of tumours? In future research, we will use a tumour-bearing animal model to explore the regulation of poly(I:C) on local immune composition and functional activity of cervical cancer and related mechanisms.

CO N FLI C T O F I NTE R E S T
All the authors declare no conflict of interest.

AUTH O R S ' CO NTR I B UTI O N
XL, HW and XY designed these experiments. XL, QS, BW, XY and LC involved in performing the experiment and analysed the data. XL and LM contributed to manuscript preparation and wrote the manuscript. XL and XY revised the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used to support the findings of this study are available from the corresponding author upon request.