Polarization of ILC2s in peripheral blood might contribute to immunosuppressive microenvironment in patients with gastric cancer

Abstract

Newly identified nuocytes or group 2 innate lymphoid cells (ILC2s) play an important role in Th2 cell mediated immunity such as protective immune responses to helminth parasites, allergic asthma, and chronic rhinosinusitis. However, the contributions of ILC2s in the occurrence and development of cancer remain unknown. Our previous study found that there was a predominant Th2 phenotype in patients with gastric cancer. In this study, the ILC2s related genes or molecules in PBMC from patients with gastric cancer were measured, and the potential correlation between them was analyzed. The expression levels of RORα, GATA3, T1/ST2, IL-17RB, CRTH2, IL-33, IL-5, and IL-4 mRNA were significantly increased in patients, but no significant changes were found in ICOS, CD45, and IL-13 expression, and there was a positive correlation between RORα or IL-13 and other related factors, such as ICOS and CD45. The increased frequency of ILC2s was also found in PBMC of patients by flow cytometry. In addition, the mRNA of Arg1 and iNOS were also significantly increased in patients. These results suggested that there are polarized ILC2s in gastric cancer patients which might contribute to immunosuppressive microenvironment and closely related to the upregulation of MDSCs and M2 macrophages.

Figure 1

The levels of RORα and GATA3 mRNA in PBMC. qRT-PCR analysis of RORα (a) and GATA3 (b) mRNA levels in PBMC from gastric cancer patients and healthy controls. Data shown are represented as mean ± SD (all samples were measured in triplicate). *P < 0.05 and **P < 0.01.

Figure 2

The levels of ILC2s-associated receptors and surface markers. T1/ST2, IL-17RB, CRTH2, ICOS, and CD45 mRNA in PBMC from gastric cancer patients were detected by qRT-PCR. The results of receptors analysis: T1/ST2 (a) and IL-17RB (b); the surface markers: CRTH2 (c), ICOS (d), and CD45 (e). Data shown were represented as mean ± SD (all samples were measured in triplicate). *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

Enhanced levels of type 2 cytokines in gastric cancer. The qRT-PCR analysis of IL-33 (a), IL-13 (b), IL-5 (c), and IL-4 (d) mRNA levels in PBMC from gastric cancer patients and healthy controls; ELISA analysis of IL-13 (e), IL-5 (f), IL-33 (g), and IL-25 (h) protein levels in plasma. Data shown are represented as mean ± SD (all samples were measured in triplicate). *P < 0.05 and **P < 0.01; ns means no significance.

Figure 4

Correlation between the transcription factor RORα and related genes mRNA levels. (a) The correlation of RORα and T1/ST2 mRNA expression (r = 0.7556, P < 0.0001), (b) RORα and IL-17RB (r = 0.7237, P < 0.0001), (c) RORα and CRTH2 (r = 0.7689, P < 0.0001), (d) RORα and ICOS (r = 0.5250, P = 0.0003), (e) RORα and CD45 (r = 0.5601, P < 0.0001), (f) RORα and IL-13 (r = 0.4336, P = 0.0041), and (g) RORα and IL-5 (r = 0.6273, P < 0.0001) in gastric carcinoma patients. There were positive correlations between them.

Figure 5

Enhanced ILC2s frequency in the PBMC from gastric cancer patients. The frequency of ILC2s in PBMC was analyzed by flow cytometry. (a) Representative diagrams of flow cytometry analysis for circulating ILC2s; (b) the frequency of ILC2s in PBMC from patients with gastric cancer was significantly increased compared with healthy controls (n = 30, P < 0.01).

Figure 6

Increased expression levels of Arg1 and iNOS in gastric cancer patients. qRT-PCR analysis of Arg1 (a) and iNOS (c) mRNA levels in PBMC from gastric cancer patients and healthy controls and ELISA analysis of Arg1 protein levels (b) in plasma. Data shown were represented as mean ± SD (all samples were measured in triplicate). *P < 0.05; ns means no significance.

Figure 7

Correlation analysis between IL-13 and ICOS and CD45 mRNA levels. (a) The correlation of IL-13 and ICOS mRNA expression (r = 0.5033, P = 0.0013) and (b) IL-13 and CD45 mRNA expression (r = 0.4646, P = 0.0015) in gastric cancer patients; there was a positive correlation between them, respectively.