Cancer Letters

Cancer Letters

Volume 381, Issue 1, 10 October 2016, Pages 156-164
Cancer Letters

Original Articles
Interleukin-10 attenuates tumour growth by inhibiting interleukin-6/signal transducer and activator of transcription 3 signalling in myeloid-derived suppressor cells

https://doi.org/10.1016/j.canlet.2016.07.012Get rights and content

Highlights

  • IL-10-deficiency accelerated tumour growth via the up-regulation of IL-6.

  • IL-6R blockade and STAT3 inhibitor reduced tumour growth and MDSCs.

  • STAT3 inhibitor attenuated MDSCs expansion and suppressed IL-6 overproduction in IL-10-deficient mice.

Abstract

Interleukin-10 (IL-10) is a well-characterized anti-inflammatory cytokine, but its role in anti-cancer immunity is controversial. After injection with TC-1 cancer cells, we observed more rapid tumour growth and significantly higher interleukin-6 (IL-6) production in IL-10 knockout (IL-10-/-) mice than wild-type (WT) mice. Blocking IL-6 with an anti-IL-6 receptor (IL-6R) monoclonal antibody (mAb) inhibited tumour growth and myeloid-derived suppressor cell (MDSC) generation, which were significantly increased in IL-10-deficient mice. MDSCs and tumour cells from IL-10-/- mice had increased phosphorylated signal transducer and activator of transcription 3 (p-STAT3) levels. Treatment with a STAT3 inhibitor, S3I, reduced tumour growth, inhibited MDSC expansion, reduced IL-6 in tumours, and relieved T cell suppression. The combination of anti-IL-6R mAb and S3I further inhibited tumour growth compared to S3I treatment alone. These results suggested that the inhibition of the IL-6/STAT3 signalling axis is a candidate anti-cancer strategy, especially under systemic inflammatory conditions with high IL-6.

Introduction

Myeloid-derived suppressor cells (MDSCs) are an immunosuppressive population consisting of precursors of macrophages, dendritic cells, granulocytes, and so on [1]. MDSCs expand and accumulate in pathological conditions, including infections, inflammation, and cancer [1], [2]. In the case of cancer, MDSCs, which are induced by both tumour-derived and host-derived factors, inhibit anti-tumour immune responses and contribute to the establishment of an immunosuppressive tumour environment [3], [4]. MDSCs suppress T cell proliferation and inhibit T cell function. They also induce regulatory T cell (Treg cell) activation and expansion [5]. Immune suppression of MDSCs is not restricted to T cell responses, and MDSCs downregulate natural killer (NK) cell activity [6] and dendritic cell function [7], which play critical roles in anti-tumour immunity. Therefore, the regulation of MDSCs is considered one of the most important steps in anti-tumour immunotherapy.

IL-10 is clearly indispensable in the regulation of inflammation and may be related to the immunosuppressive functions of MDSCs [8]. However, since it was discovered in the early 1990s, there is conflicting evidence regarding the precise role of IL-10 in cancer development. It has been reported that IL-10 mediates tumour-inhibitory effects in various cancer types. Although increased levels of IL-10 have been detected in biopsies and premalignant lesions of cervical cancer patients [9], decreased IL-10 levels are also associated with the risk of cervical cancer [10]. In other cancer types, a hypomorphic variant that produces low levels of IL-10 is a high risk factor for gastric adenocarcinoma [11], and low levels of IL-10 are associated with prostate cancer development [9], [12]. In several cases, IL-10 is involved in tumour promotion. For example, IL-10 expression is highly associated with the development of HPV-positive cervical cancer [13]. In a mouse model of tumour transplantation, tumour-infiltrating macrophages produce IL-10, which might promote the induction of Treg cells and MDSCs as immune evasion mechanisms to facilitate tumour growth [14].

The tumour-inhibitory effect of IL-10 might be related to its ability to modulate other cytokines or pro-tumoural mediators. IL-10 inhibits tumour growth via the inhibition of several inflammatory and angiogenic factors, including vascular endothelial growth factor, interleukin-1β (IL-1β), TNF-α, and IL-6 [11]. Blocking IL-1β ameliorates tumour growth enhanced by IL-10 deficiency [15]. Unexpectedly, IL-10 inhibits the production of inflammatory cytokines and dampens the generation of MDSCs as well as Treg cells in the tumour environment, and the depletion of MDSCs restores tumour growth in IL-10-deficient conditions [15]. Lipopolysaccharide-stimulation increases the production of IL-1α and IL-1β by MDSCs, and IL-1 blockade by IL-1 receptor α (IL-1Rα) efficiently inhibits tumour growth in IL-10-deficient conditions.

Similar to IL-10 deficiencies, IL-1R-deficient mice exhibit delayed accumulation of MDSCs after tumour transplantation and show reduced tumour growth with decreased potential for inflammation [16]. Interestingly, the decreased accumulation of MDSCs and tumour progression in IL-1R-deficient mice are restored via the additional expression of IL-6 by tumour cells, suggesting that IL-6 is a downstream mediator of IL-1R signalling [16]. In addition, the decreased IL-6 production and reduced expression of p-STAT3 in C/EBP-homologous protein (CHOP)-deficient MDSCs may be associated with the decreased suppressive function of MDSCs, conferring antitumour immunity to T cells [17]. IL-6 overexpression on cancer cells restores the immune-suppressive activity of CHOP-deficient MDSCs. Based on these previous reports, we inferred that IL-6R is a plausible target to restrict the MDSC expansion observed in IL-10-deficient mice.

In the current study, we examined the mechanisms of increased tumour growth in IL-10-deficient mice. We adopted a TC-1 cervical cancer model with subcutaneously (s.c.) transplanted cancer cells. Based on our initial finding that IL-10-deficient mice with TC-1 tumour transplantation produced significantly higher levels of IL-6, we administered anti-IL-6R monoclonal antibodies (mAbs) to tumour-bearing mice and found that IL-6R blockade efficiently slowed TC-1 tumour growth in wild-type (WT) and IL-10-deficient mice. The levels of p-STAT3 in MDSCs from IL-10-deficient mice were significantly higher than those from control mice, and were decreased by the IL-6R blockade. Treatment of mice with a STAT3 inhibitor (S3I) dampened the production of IL-6 in tumour tissues, and co-treatment with the IL-6R blockade and STAT3 inhibitor had greater anti-tumour effects than S3I treatment alone in IL-10-deficient mice. Collectively, these data suggest that IL-10 attenuate tumour growth by inhibiting IL-6/STAT3 axis on MDSCs.

Section snippets

Mice

All experiments were approved by the Institutional Animal Care and Use Committee of Kangwon National University. C57BL/6 mice were purchased from Charles River Laboratories (Orient Bio Inc., Sungnam, Korea). IL-10-/- mice (C57BL/6 background) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were bred and housed in specific pathogen-free conditions, and IL-10-/- mice and their littermate controls were used for tumour inoculation. All mice used in the experiments were 6–8

IL-10-deficiency accelerated tumour growth via the upregulation of IL-6 in mice

Several previous studies have suggested that tumour growth is significantly more rapid in IL-10-/- mice than WT mice [15], [20]. To confirm this pattern for TC-1 cervical cancer cells expressing HPV E6 and E7, we s.c. injected 5 × 105 TC-1 cells/mouse into the left flank of WT and IL-10-/- mice and measured tumour volume over a 5-week period. TC-1 tumour growth was significantly faster in IL-10-/- mice than in WT mice (Fig. 1A). Based on a previous report showing that IL-6 levels are highly

Discussion

IL-10 is produced by most leukocytes, including myeloid and lymphoid cells. Several subsets of T cells are important sources of IL-10 [31]. The receptor of IL-10 is most highly expressed on macrophages and dendritic cells. An IL-10 deficiency in mice is associated with various inflammatory diseases, such as bone abnormalities, inflammatory bowel disease, and other metabolic diseases [21], [32]. In addition, low levels of IL-10 in cancer are a risk factor for prostatic cancer, gastric

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2014R1A2A2A01002576) of Korea. This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI15C0450).

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgements

The authors thank Chugai Pharmaceutical Co., Ltd. for the gift of the MR16-1 (anti-IL-6R mAb), and thank Prof. Tae Woo Kim for sharing TC-1 cell.

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