Increased IL-17 production correlates with immunosuppression involving myeloid-derived suppressor cells and nutritional impairment in patients with various gastrointestinal cancers

Although a causal relationship between inflammation and innate immunity of cancer is more widely accepted today, many of the precise cell mechanisms mediating this relationship have not been elucidated. Th17 cells, which produce the proinflammatory cytokine interleukin 17 (IL-17), have been recognized as one of the key factors in the regulation of inflammatory bowel disease and rheumatoid arthritis. This study demonstrated that, in patients with various types of gastrointestinal cancer, IL-17 production was correlated with myeloid-derived suppressor cell (MDSC) levels and with markers for nutritional impairment, immune suppression and chronic inflammation. IL-17 was significantly higher in patients with various types of gastrointestinal cancer compared to normal volunteers. In addition, IL-17 levels were significantly correlated with neutrophil counts and the neutrophil/lymphocyte ratio (NLR) and significantly inversely correlated with cell-mediated immune response indicators [lymphocyte phytohemagglutinin (PHA)-blastogenesis and IL-12 induction] and patient nutritional status (prealbumin levels). Circulating MDSC levels were significantly correlated with IL-17 production. These results suggest that, in human gastrointestinal cancers, chronic inflammation involving IL-17 may be an important mechanism contributing to disease progression through enhancement of immune suppression or cachexia. Controlling the activation of Th17 cells may prove to be a valuable strategy for the treatment of gastrointestinal cancer patients.


Introduction
To the best of our knowledge, the functional relationship between inflammation and cancer has not been recently investigated. Karl Virchow hypothesized that cancer originates at sites of chronic inflammation (1). Although a causal relationship for inflammation and innate immunity of cancer is more widely accepted today, the precise cell mechanisms mediating this relationship have not been elucidated. Th17 cells were identified in 2005 (2)(3)(4) and in humans, the cytokines that direct Th17 cell lineage development likely include IL-6, IL-21, IL-23 and IL-1-β. In addition, TGF-β plays a potentially synergistic role through its ability to suppress Th1 cell lineage commitment (5,6). Although the IL-17 cytokine family includes six members, Th17 cells are considered to produce only the proinflammatory cytokines IL-17A and IL-17F, which are 55% identical. IL-17A and IL-17F combine to form a heterodimer (7). It was previously reported that IL-17 plays an important role in the pathogenesis of inflammatory bowel diseases (IBDs), including Crohn's disease and ulcerative colitis (8,9).
Myeloid-derived suppressor cells (MDSCs) have been identified in the majority of patients and experimental mice with tumours and inflammation, based on their ability to suppress T-cell activation (10). In mice, MDSCs are uniformly characterised by expression of the cell surface molecules detected by antibodies to Gr1 and CD11b (11). Variations in the MDSC phenotype are consistent with the concept that MDSCs are a diverse family of cells that are in various intermediate stages of myeloid cell differentiation.
In humans, MDSCs are most commonly defined as CD14 -CD11b + cells or, more narrowly, as cells that express the common myeloid marker CD33 but not the markers of mature myeloid or lymphoid cells, or the MHC class II molecule HLA-DR (12). For the purposes of this study, MDSCs were defined as CD14 -CD11b + CD33 cells.
Tumour development and growth occurs as a result of interactions between tumour and host immune/inflammatory

Increased IL-17 production correlates with immunosuppression involving myeloid-derived suppressor cells and nutritional impairment in patients with various gastrointestinal cancers
cells and chronic inflammation plays an important role in cancer development and progression (13,14). Inflammatory parameters based on differential white cell counts, such as the neutrophil̸lymphocyte ratio (NLR), may be simple and readily available biomarkers for tracking inflammation and cancer development. The results of the present study demonstrate the correlation of IL-17 production levels with MDSCs and other markers for nutritional status, immune suppression and chronic inflammation in patients with a variety of gastrointestinal cancers.   Cytokine production by PBMCs. Samples (20 ml) of blood collected directly from heparinized collection tubes were subjected to Ficoll-density gradient centrifugation in order to isolate the PBMCs, 10 6 of which were incubated in 1 ml of RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL, Rockville, MD, USA) and 20 µg/ml phytohemagglutinin (PHA) (Sigma, St. Louis, MO, USA) in 5% CO 2 at 37˚C for 24 h. Aliquots of these supernatants were then frozen and maintained at -80˚C until use. Supernatant samples subsequently were thawed and used for measurement of IL-17 and IL-12 concentrations using ELISA test kits (R&D Systems, Minneapolis, MN, USA). Each sample was used only once after thawing.

Materials and methods
Lymphocyte proliferation assay. Lymphocyte proliferation assays were performed using PBMC suspended in RPMI-1640 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) containing 10% fetal calf serum (Sigma). Following the addition of 10 µg/ml PHA into PBMC culture wells kept at 37˚C in a  H-thymidine (Japan Radioisotope Association, Tokyo, Japan) was added to the wells for the last 8 h of incubation. Cells were harvested and 3 H-thymidine incorporation was determined using a liquid scintillation counter (PerkinElmer, Inc., Waltham, MA, USA) and expressed as counts per minute (cpm). The stimulation index (SI) was obtained by calculating total cpm/control cpm. The controls were defined as PBMCs that had not been subjected to PHA addition.

Markers for nutritional status and chronic inflammation.
To evaluate the nutritional status of the subjects, serum concentrations of albumin (determined by nephelometry) and prealbumin (determined using a turbidimetric immunoassay) were measured using standard protocols. Neutrophil and lymphocyte counts, as well as their ratios (NLR) in peripheral blood samples, were used as indicators of inflammation in this study.
Statistical analysis. Differences between groups were determined by Student's t-tests. Correlations between two variables were quantified by determining the Spearman's rank correlation coefficients. P<0.05 was considered to indicate a statistically significant difference. Inadequate amounts of blood were obtained from some patients and in these cases, certain measurements were not possible.     (Fig. 4). The production of IL-17 demonstrated a significant inverse correlation with SIs (found by assessing lymphocyte PHA-blastogenesis) (P<0.05, r=-0.302) and IL-12 production (P<0.01, r=-0.411), as well as a significant positive correlation with circulating levels of MDSCs (P<0.001, r=0.492) (Fig. 5).

Discussion
To the best of our knowledge, this study is the first to describe an important role for IL-17 in the induction of immune suppression and nutritional impairment during systemic inflammation. IL-17 production was significantly higher in patients with various types of gastrointestinal cancer compared to that in normal volunteers. Production levels were significantly correlated with neutrophil counts and NLRs. By contrast, they were significantly inversely correlated with cell-mediated immune responses, including lymphocyte PHA-blastogenesis and Th1 induction, as reflected by IL-12 production and compromised patient nutritional status, as reflected by prealbumin levels. Circulating levels of MDSCs were also significantly correlated with IL-17 production levels.
Over the last decade, there has been an expansion of scientific knowledge regarding the pathogenesis of IBDs, including Crohn's disease and ulcerative colitis. IBDs have been reported to arise due to a combination of genetic variations and alterations in intestinal microflora, which may subsequently promote an uncontrolled immune response and result in chronic intestinal inflammation (8,9). IL-17 is considered to stimulate various types of cells to produce proinflammatory mediators that amplify intestinal inflammation. It has also been reported that in humans, Th17 cells play an essential role in protective immunity against certain microorganisms (15). Th17 cells have a close developmental link with FOXP3 + CD4 + regulatory T cells. Th17 cells have been reported to transiently express FOXP3 during their development (16). In the present study, the inflammation induced by IL-17 appeared to cause the production of MDSCs that may potentially inhibit maturation of dendritic cells, and cell-mediated immunity may be suppressed through Th2 dominant conditions driven by the depressed production of IL-12. Thus, Th17 cells may play important roles in the development of immune suppression in patients with malignant diseases.
The NLR was reported to be a marker of systemic inflammatory response and an independent predictor of clinical benefit, good prognosis and survival in patients receiving cancer chemotherapy (17). The likelihood of the association of IL-17 production with nutritional impairment is high, due to the role of IL-17 as a marker of systemic inflammation. It was previously reported that the key mechanisms leading to cancer cachexia, in which nutritional impairment is a major clinical issue, are mostly immune reactions caused by chronic inflammation and that treatment with a COX-2 inhibitor or a specific nutrient formula is effective (18,19).
Thus, in human cancers, chronic inflammation involving IL-17 is considered to be important in the development of disease-advancement indicators, such as immune suppression  A B C or cachexia. In order to suppress Th17, several candidates have been experimentally used. Since it was reported that Th17 cells are induced by TGF-β, IL-1, IL-6, IL-21 or IL-23, antibodies targeting IL-6 or IL-23 have been considered strong candidates for the development as treatments for autoimmune diseases, including IBD and rheumatoid arthritis (1,20). Further investigations are required to explore this possibility and gain more insight into this field of medicine.