Introduction
Lung cancer (LC) is currently the leading cause of cancer-related mortality among both males and females worldwide and was responsible for approximately 1.59 million deaths in 2012 (1,2). LC has also been the number one cause of cancer-related deaths among patients with malignant tumors since 2008 in China (3). In addition, the mortality rate of LC in China has increased dramatically during the past three decades due to smoking and air pollution, imposing a huge economic burden on patients, medical professionals, and society (3).
LC is characterized by a series of hallmarks, such as tumor-promoting inflammation, avoidance of immune destruction, genomic instability, and induction of angiogenesis (4). Inflammatory responses contribute to the initiation, progression, and metastases of malignancies as proposed by Virchow in 1863, by promoting proliferative signaling, destabilizing genomic integrity, and inducing the invasion of cancer cells (5,6). Chronic inflammation triggered by bacterial and viral infections, tobacco smoking, and chemicals suppress wound healing and tissue regenerative responses, promoting cancer development and progression, as described as 'wounds that do not heal' (7,8). In addition, inflammation compromises genomic maintenance and repair pathways, inducing genomic instability (6,9). Furthermore, cancer cells modulate inflammation by secreting soluble mediators and interfering with innate and adaptive immune cells, such as, macrophages, dendritic cells and lymphocytes (6). Multiple inflammatory mediators may trigger and maintain tumorigenesis individually or coordinately in the tumor microenvironment (6). The crosstalk between the inflammatory microenvironment and cancer cells controls and shapes tumor growth and metastasis (5–7).
Interleukin (IL)-17A, a proinflammatory cytokine discovered in 1993, induces tissue inflammation mainly by promoting expression of various cytokines, chemokines, antimicrobial peptides, and tissue-remodeling molecules (10–12). IL-17A exerts complicated functions in allergic, autoimmune, and malignant diseases, by targeting mesenchymal and myeloid cells (10,13). Although originally linked to IL-17-producing CD4+ T helper (Th17) cells, a distinct T cell subtype different from Th1 and Th2 cells, IL-17A was subsequently found to be produced by several other immune cells, including IL-17A-producing γδT (γδT17) cells, IL-17A-producing CD8+ T (Tc17) cells, natural killer T cells, and mast cells (10,14,15). IL-17A-producing T cells and associated cytokines, such as IL-17, IL-23, and IL-1β, have been shown to be involved in both inflammation and immune responses in various types of cancers including gastric, breast, prostate and hepatocellular cancer (10,16–19). However, the roles of these T cells and associated cytokines are conflicting in various animal models and patients (10,20–22). IL-17-producing T cells have displayed both antitumor and protumor functions, due to their plasticity and functions in the tumor microenvironments (23). The numbers of circulating Th17 and γδT17 cells were significantly higher in patients with gastric cancer than those in healthy controls (HCs) (16). On the contrary, data have shown that Th17 cells elicit antitumor effects, by promoting cytotoxic activities, enhancing Th1 response, and augmenting the expression of MHC antigens (24–26). In a murine model of LC, enhanced Th17 cells and overexpression of IL-17A stimulated tumor growth in the lungs (27,28). Similarly, an increased number of intratumoral IL-17-positive cells in patients with LC was correlated with poor prognosis (29). However, a higher percentage of Tregs but a lower frequency of Th17 cells was found in malignant pleural effusion, as compared with those in parapneumonic effusion, and a higher ratio of Treg/Th17 cells in malignant pleural effusion was found to indicate a poor prognosis of patients with LC (30). Thus, the roles of IL-17A-producing T cells, especially Tc17 and γδT17 cells, and associated cytokines in the progression of LC remain to be defined.
In the present study, we investigated the frequencies of IL-17-producing T cells (Th17, Tc17 and γδT17 cells) and levels of IL-17A-associated cytokines (IL-17A, IL-23, IL-1β, and TGF-β1) in patients with lung adenocarcinoma (LA) and HCs. We found that the frequencies of both Th17 and γδT17 cells in the peripheral blood of patients with LA were higher than those in HCs and were positively associated with tumor invasion and metastasis, whereas the frequency of Tc17 cells in patients with LA was decreased. Furthermore, the major source of IL-17A production was Th17 cells in peripheral blood from both patients with LA and HCs. In addition, the protein and corresponding mRNA levels of IL-17A, IL-23, IL-1β, and TGF-β1 were much higher in patients with LA than those in HCs, and the levels of IL-17A were positively correlated with numbers of both Th17 and γδT17 cells, but not Tc17 cells. Finally, the frequencies of circulating Th17 and γδT17 cells, along with levels of IL-17A, IL-23, IL-1β, and TGF-β1 were decreased in the patients with LA after tumor resection, whereas the frequency of circulating Tc17 cells was inversely increased in these patients. This study provides further insight into the association between IL-17A-producing T cells and progression of LA, and offers promising targets for therapeutic strategies.
Materials and methods
Subjects
Forty patients, diagnosed with LA and admitted to the Department of Respiratory Medicine at the First Affiliated Hospital of Zhejiang University, and 35 HCs were enrolled in this study from May 2014 to June 2015. All patients were histologically confirmed with LA by two pathologists. The patients were excluded if they had autoimmune diseases, immune compromised diseases, and pulmonary infections, if they were taking any drugs which affect immune responses, or if they had already received anticancer therapies including chemotherapy, radiotherapy, targeted therapy, surgery or immune therapy. The characteristics of the enrolled 40 patients and 35 HCs, are summarized in Table I. All cases with LA were staged according to the 7th edition of the tumor, node, and metastasis (TNM) classification for LC (31). The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University, and informed consent was obtained from all patients and HCs.
Sample collection and processing
Peripheral blood samples were collected from the subjects before any regional or systemic anticancer treatments and re-collected from 15 patients after thoracic surgery. The fresh peripheral blood of all individuals was stored in heparin-coated tubes (BD Biosciences, San Jose, CA, USA) and centrifuged at 4,000 rpm for 10 min at 4°C. Then, the cell-free supernatants, allocated into 1.5-ml Eppendorf tubes, were frozen at −80°C for the detection of cytokines (30). The cell pellets were re-suspended in saline for further analyses of flow cytometry and real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (30).
Flow cytometric analysis
Human peripheral blood mononuclear cells (PBMCs), isolated from cell pellets using Ficoll-Hypaque density gradient centrifugation, were re-suspended in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen Life Technologies, Carlsbad, CA, USA). Next, they were stimulated for 5 h with 50 ng/ml phorbol 12-myristate 13-acetate (PMA; BioVision, Mountain View, CA, USA), 1 µg/ml ionomycin (Enzo Life Sciences, Inc., Farmingdale, NY, USA) and 500 ng/ml monensin (eBioscience, San Diego, CA, USA) in 24-well plates (16). To analyze IL-17A-producing T cells, stimulated PBMCs were stained with phycoerythrin (PE)-conjugated anti-human CD3, fluorescein isothiocyanate (FITC)-conjugated anti-human γδTCR, allophycocyanin (APC)-conjugated anti-human CD8, and Pacific Blue-conjugated anti-human CD4 antibodies at 4°C for 30 min (16). Then, the cells, fixed and permeabilized with IC fixation/permeabilization buffer (eBioscience), were intracellularly stained with PerCP-Cy5.5-conjugated anti-human IL-17 antibody according to the manufacturer's instructions (16). All antibodies used in the flow cytometric analysis were obtained from Biolegend (San Diego, CA, USA) and isotype-matched antibody controls were used in all procedures. Flow cytometric acquisition was performed using a FACSCalibur (BD Biosciences), and data were analyzed using FlowJo software, version 7.6.5 (TreeStar, Inc., San Carlos, CA, USA).
ELISA measurement of serum cytokines
The cell-free supernatants of all individuals were tested using ELISA kits according to the manufacturer's instructions (eBioscience), for cytokines including IL-17A, IL-23, IL-1β, and TGF-β1. All samples were tested in triplicate.
qRT-PCR
RNA samples were prepared from stimulated human PBMCs using TRIzol (Invitrogen Life Technologies) (16). cDNA was synthesized using reverse transcription reagent kits (Takara Biotechnolgy Co., Inc., Dalian, China) and real-time PCR was performed in triplicate using the QuantiFast™ SYBR Green PCR kit (Qiagen, Hilden, Germany) in an ABI 7500 analysis system (Applied Biosystems, Foster City, CA, USA) (16). The following primer pairs were used: IL-1β forward, 5′-CCACAGACCTTCCAGGAGAATG-3′, and reverse, 5′-GTGCAGTTCAGTGATCGTACAGG-3′; IL-17A forward, 5′-CGGACTGTGATGGTCAACCTGA-3′, and reverse, 5′-GCACTTTGCCTCCCAGATCACA-3′; IL-23p19 forward, 5′-GAGCCTTCTCTGCTCCCTGATA-3′, and reverse, 5′-GACTGAGGCTTGGAATCTGCTG-3′; TGF-β1 forward, 5′-CAGAAATACAGCAACAATTCCTGG-3′, and reverse, 5′-TTGCAGTGTGTTATCCGTGCTGTC-3′; GAPDH forward, 5′-GGTCTCCTCTGACTTCAACA-3′, and reverse, 5′-GTGAGGGTCTCTCTCTTCCT-3′. The data were analyzed by ABI 7500 software (Applied Biosystems).
Statistical analysis
Values are presented as means ± SEM. Differences among groups were tested by one-way ANOVA. Differences between two groups were tested using non-paired Student's t-test. For non-parametric data, the Mann-Whitney U test was performed between groups. Correlations between values were determined using Spearman's correlation coefficient. Analysis was performed with SPSS statistical software (version 21.0; SPSS, Inc., Chicago, IL, USA), and P<0.05 was considered statistically significant.
Discussion
Evidence has shown the dual roles of T cells and associated cytokines in the initiation, progression, and metastasis of LC. The present study investigated the frequencies of IL-17A-producing cells (Th17, Tc17, and γδT17 cells), and expression levels of IL-17-associated cytokines in patients with LA and HCs. We found that the frequencies of circulating Th17 and γδT17 cells, and the protein and corresponding mRNA levels of IL-17A, IL-23, IL-1β, and TGF-β1 in the peripheral blood of patients with LA were higher than those in the HCs, whereas the frequency of Tc17 cells in patients with LA was decreased. Moreover, the frequencies of circulating Th17 and γδT17 cells, along with the levels of IL-17A, IL-23, IL-1β, and TGF-β1 were decreased in patients with LA after tumor resection. Our findings suggest that Th17, γδT17, Tc17 cells, and IL-17A-associated cytokines contribute to the development of LA and thus represent promising targets for therapeutic strategies.
Th17 cells were originally discovered to promote inflammatory responses in autoimmune diseases and act in host defenses against microbes (32,33). Recently, their functions in the initiation and development of cancers have been extensively studied (34). However, the roles of Th17 cells and its main cytokine, IL-17A, in carcinogenesis are still controversial (35). Accumulation of tumor-infiltrating Th17 cells have been found in a variety of cancers, including gastric, hepatocellular, breast, and LCs, and contributed to poor patient prognosis (28,36–38). Cytokines secreted by cancer cells, such as RANTES, MCP1, IL-6, IL-1β, and CCL 20, contribute to the differentiation and expansion of Th17 cells, in the tumor microenvironment (35,39). Moreover, Th17 cells and IL-17 resulted in the recruitment of Gr-1+CD11b+ myeloid cells, promoting the inflammation and tumor growth in a mouse model with oncogenic K-ras mutation expressed only in the lungs (28). Smoking-mediated Th17 inflammation by induction of osteopontin and IL-17A deficiency attenuated smoking-induced emphysema in mice (40). In contrast, elevated Th17 cells or a high ratio of Th17/Treg, in malignant pleural effusion, partially promoted by chemokines, predicted a good prognosis in patients with LC (30,41). Th17 cells may promote the recruitment of dendritic cells and subsequent activation of CD8+ T cells in tumors, exerting antitumor immunity (26). In IL-17-deficient mice, tumor growth and lung metastasis were augmented, accompanied by decreased natural killer cells and T cells, suggesting the protective roles of endogenous IL-17 and Th17 in tumors (25). Consistent with previous studies, our results demonstrated that the frequency of circulating Th17 cells was increased in the patients with LA, and was associated with tumor invasion and metastasis, indicating the pro-tumoral roles of Th17 cells in the initiation and progression of LA (42). It was also confirmed that Th17 cells were the main source of IL-17A in human peripheral blood in both patients with LA and HCs. However, the mechanisms involved in the accumulation of Th17 cells in the tumor microenvironment are still unknown, and Th17 cells could be induced, expanded, or converted from other T cells.
Tc17 cells, a minor subset of CD8+ T cells characterized by the production of IL-17, play various roles in infection, cancers and autoimmune diseases (43,44). Although most of the knowledge concerning the differentiation and plasticity of Tc17 cells stems from autoimmune diseases, emerging evidence has also shown that Tc17 cells are associated with the development of cancers in both animal models and humans (43). In patients with hepatocellular carcinoma, tumor-activated monocytes triggered the proliferation of Tc17 cells at the edge of invading tumors, by a set of proinflammatory cytokines (45). In patients with uterine cervical cancer, high levels of Tc17 cells were found in both the peripheral blood and in cervical tissues, and were involved in the metastases of pelvic lymph nodes and tumor vasculogenesis (46). Notably, adoptive transfer of tumor-specific Tc17 cells led to the regression of melanoma in mice by the recruitment of neutrophils and induction of chemokines, and elicited antitumor immunity, which was reversed by the blocking of the inducible costimulator pathway (47–49). The numbers of both CD4+ and CD8+ T cells were significantly elevated in lung tumor tissues compared with HCs, and were positively related to the staging of cancers (50). In spite of some functional similarities shared with Th17 cells, it has become gradually clear that Tc17 cells are a distinct subset of T cells regarding differentiation, development, and plasticity. Contrary to Th17 cells, the frequency of circulating Tc17 cells was reduced in the patients with LA, especially those with tumor invasion and distant metastasis. These results are consistent with data from patients with thyroid tumors or gastric carcinoma, suggesting that Tc17 cells may exhibit some antitumor activities in lung carcinoma and Th17 cells may suppress the development of Tc17 in the tumor microenvironment (16,51).
γδT cells, compared with conventional αβT cells, exhibit distinct and versatile functions by recognition of non-peptide antigens, production of proinflammatory cytokines (IL-17, interferon-γ, and TNF-α), and interaction with activation of adaptive immune cells (21,52). γδT17 cells, the primary source of IL-17 in the early stage of some diseases and one of the pivotal players in immune surveillance, display diverse responses to tumors (20,53). In patients with colorectal cancers, γδT17 cells were found to be a major source of IL-17, related to the expansion of myeloid-derived suppressor cells, and positively associated with tumor stages (54). Murine γδT17 cells also mobilized small peripheral macrophages, which expressed proangiogenic and proinflammatory mediators, and promoted ovarian cancer growth in vivo (55). Recently, in a murine model of breast cancer, γδT17 cells resulted in expansion and polarization of specific neutrophils which subsequently inhibited cytotoxic CD8+ lymphocytes, and led to pulmonary and lymph nodal metastases, indicating a cooperative mechanism among γδT17 cells, cytotoxic T cells and neutrophils in the metastatic microenvironment (18). In our study, γδT17 cells were the third source of IL-17A, which were consistent with gastric patients but not with colorectal cancers (16,54). In addition, increased frequency of γδT17 cells was found in patients with LA and was positively related to the metastasis and staging of cancers, and was markedly decreased after the resection of the tumor. The prevalence and variety of γδT17 cells in patients with LA were very similar with those of Th17 cells, suggesting these two IL17-producting T cells may collaboratively promote pulmonary carcinogenesis.
In response to stress, injury, and pathogenic stimuli, IL-17-associated cytokines, including IL-23, IL-1β, and TGF-β1, drive the differentiation of naïve T cells into IL-17-producing T cells (14,43,56). IL-23 further induces the production of IL-17 by Th17 and γδT cells, and promotes tumor growth (22,57). IL-17 targets myeloid and mesenchymal cells, and induces tissue inflammation by promoting the expression of proinflammatory cytokines, chemokines, and antimicrobial peptides (10). In addition, IL-17 resulted in the infiltration of myeloid-derived suppressor cells and angiogenesis in tumors, and contributes to the tumor-promoting microenvironments in mice (58,59). Elevated levels of IL-17 were found in patients with gastric, colorectal and prostatic cancers, and are associated with poor prognosis (60). Recently, increased levels of IL-23, IL-1β, and IL-17A were found in gastric patients and were positively related to tumor invasion and metastasis (24). In experimental silicosis, IL-17A produced by both Th17 and γδT17 cells was required for acute pulmonary inflammation and injury, but not chronic responses and fibrosis (61). Our study showed that both the mRNA and protein levels of IL-17A IL-23, IL-1β, and TGF-β1 in PBMCs of patients with LA were markedly higher than those in the HCs. In addition, the expression of IL-17A in serum was positively associated with the number of Th17 and γδT17 cells, but not Tc17 cells. Results indicated that these inflammatory cytokines contribute to the proliferation of Th17 and γδT17 cells, and the progression of LC in the tumor microenvironment.
We further explored the effects of the resection of lung tumors on the alterations of IL-17-producing T cells and inflammatory cytokines. Notably, after surgery in patients with LA, the frequencies of Th17 and γδT17 cells, and cytokines including IL-17A IL-23, IL-1β, and TGF-β1 were markedly reduced, whereas the frequency of Tc17 cells recovered, suggesting that removal of tumors may restore immune hemostasis and surveillance, and IL-17-producing cells may be critical to tumor progression.
In conclusion, our data demonstrated that the frequencies of circulating Th17 and γδT17 cells, and the protein and corresponding mRNA levels of IL-17A, IL-23, IL-1β, and TGF-β1 in the peripheral blood of patients with LA were higher than those in HCs, whereas the frequency of Tc17 cells in patients with LA was decreased. In addition, Th17 cells were the major source of IL-17A in patients with LA and HCs. Moreover, the frequencies of circulating Th17 and γδT17 cells, along with levels of IL-17A, IL-23, IL-1β, and TGF-β1 were decreased in the patients with LA after tumor resection. Our data suggest that Th17, γδT17 and Tc17 cells, and IL-17A-associated cytokines play pivotal roles in the crosstalk between tumor-related inflammation and immunity, and contribute to the development of LC. In the future, a better understanding of the distribution and cooperation among IL-17-producing T cells would provide us with a rationale for novel anticancer strategies.