Mast cells mobilize myeloid-derived suppressor cells and Treg cells in tumor microenvironment via IL-17 pathway in murine hepatocarcinoma model

Abstract

Tumor immunosuppression is commonly braided with chronic inflammation during tumor development. However, the relationship between immunosuppression and inflammation in tumor microenvironment is still unclear. We have demonstrated that mast cells are accumulated and exacerbate the inflammation and immunosuppression in tumor microenvironment via SCF/c-kit signaling pathway. Here, we further elucidate the underlying mechanism, which involves both myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our data showed that mast cells mobilized the infiltration of MDSCs to tumor and induced the production of IL-17 by MDSCs; MDSCs-derived IL-17 indirectly attracted Treg cells, enhanced their suppressor function, and induced the IL-9 production by Treg cells; in turn, IL-9 strengthened the survival and protumor effect of mast cells in tumor microenvironment. Our findings disclose a closed loop among mast cells, MDSCs and Treg cells in tumor microenvironment, which provides a new insight into the paralleled developments of inflammation and immunosuppression in tumor microenvironment. Based on these findings, we propose that targeting tumor inflammation might be a potential strategy to reverse the immunosuppression of tumor microenvironment, thus facilitating cancer immunotherapy.

Figure 1. The regulation of tumor-infiltrating MDSCs by mast cells.

(A) Mast cells promoted the infiltration of MDSCs into tumor microenvironment. 5×10⁶ BMMCs with or without anti-SCF or c-kit antibodies, were injected into tumor-bearing mice by i.v. injection. Bone marrow cells were used as control. Seven days later, the tumor-infiltrating lymphocytes were used to analyze Gr-1⁺CD11b⁺ MDSCs by flow cytometry. The left shown was the representative of FACS profiles. The right shown was the combined reproducible data (n = 6). *, P<0.05, compared to control. (B) Mast cells promoted the suppressive function of MDSCs in tumor microenvironment. BMMCs with or without antibodies, were injected into tumor-bearing mice (n = 6). Seven days later, tumor-infiltrating MDSCs were isolated as described in Materials and Methods and the suppression assay was performed as described in Materials and Methods . (C and D) Mast cells upregulated the expressions of CCL2, IL-10 and IL-13 in tumor microenvironment. BMMCs were injected into tumor-bearing mice. Seven days later, tumor tissues were used to analyze CCL2, IL-10 and IL-13 expressions by real time RT-PCR (C) and ELISA (D). (E) The regulation of arginase 1 by mast cells. Lane 1–2: tumor tissues from BMMC group or control were used to analyze arginase 1 expression by real time-RT-PCR and western blot. Lane 3–5: the cultured MDSCs were treated with PBS or IL-10 (20 ng/ml) or IL-13 (20 ng/ml) for 48 h, and then used for the analysis of arginase 1 expression.

Figure 2. Mast cells regulate MDSCs through IL-17 pathway.

(A) Blockade of IL-17 prevented mast cell-mediated MDSC infiltration to tumor. 5×10⁶ BMMCs were injected into tumor-bearing mice by i.v. injection. IL-17 neutralizing antibody was i.p. injected to the mice 1 h, 2 days and 5 days after BMMCs injection. On day 7, the tumor-infiltrating lymphocytes were used to analyze Gr-1⁺CD11b⁺ MDSCs by flow cytometry (left). In addition, IL-17 neutralizing antibody was i.p. injected to the mice 24 h and 1 h before BMMCs injection. 2×10⁶ CFSE-labeled MDSCs were injected into the mice two days later. The tumor tissues were surgically excised, and frozen sections were prepared and analyzed by fluorescence microscopy (right). (B) Blockade of IL-17 attenuated mast cell-mediated MDSC suppressive function. BMMCs were injected into tumor-bearing mice. IL-17 neutralizing antibody was i.p. injected to the mice at different time points. On day 7, tumor-infiltrating MDSCs were isolated for the suppression assay. (C) IL-17 was not expressed by H22 tumor cells, T cells or B cells. BMMCs were injected into tumor-bearing mice. Seven days later, tumor cells and TILs were isolated, respectively. The expression of IL-17 was analyzed by flow cytometry. (D and E) Mast cells upregulated the expression of IL-17 by MDSCs. Seven days after BMMCs injection, the isolated TILs were used for IL-17 expression analysis. The data showed the upregulation of IL-17 by CD11b⁺ cells in BMMC group (D), and most of the gated IL-17⁺ cells expressed Gr-1 marker (E).

Figure 3. Mast cells regulate the infiltration and function of Treg cells in tumor microenvironment.

(A) Mast cells regulate the infiltration of Treg cells. 5×10⁶ BMMCs with or without anti-SCF or c-kit antibodies, were injected into tumor-bearing mice by i.v. injection. Bone marrow cells were used as control. Seven days later, the tumor-infiltrating lymphocytes were used to analyze CD3⁺Foxp3⁺ Treg cells by flow cytometry. The left shown was the representative of FACS profiles. The right shown was the combined reproducible data (n = 6), *, P<0.05, compared to BM cell control. (B) Mast cells regulate the suppressive function of Treg cells. BMMCs with or without antibodies, were injected into tumor-bearing mice (n = 6). Seven days later, tumor-infiltrating Treg cells were isolated as described in Materials and Methods and the suppression assay was performed as described in Materials and Methods .

Figure 4. Mast cell-induced IL-17 mediates Treg cell infiltration via upregulating chemokines CCL17 and CCL22.

(A) The interference of IL-17 impaired the effect of mast cells on Treg cell infiltration. 5×10⁶ BMMCs were injected into tumor-bearing mice by i.v. injection. IL-17 or CCL2 neutralizing antibody or Gr-1 depleting antibody was i.p. injected to the mice 1 h, 2 days and 5 days after BMMCs injection. On day 7, the tumor-infiltrating lymphocytes were isolated to analyze CD3⁺Foxp3⁺ Treg cells by flow cytometry. The results were combined from three mice. (B and C) Mast cells-induced IL-17 upregulated CCL17 and CCL22 expressions in tumor microenvironment. BMMCs were injected into tumor-bearing mice. IL-17 or Gr-1 antibody was i.p. injected to the mice at different time points. Seven days after BMMCs injection, the tumor tissues were used to analyze CCL17 and CCL22 expressions by real time RT-PCR (B) and ELISA (C). (D) The effect of CCL17 and CCL22 on Treg cell infiltration. BMMCs were injected into tumor-bearing mice. CCL17 or CCL22 neutralizing antibody was i.p. injected to the mice 1 h, 2 days and 5 days after BMMCs injection. On day 7, the tumor-infiltrating lymphocytes were isolated to analyze CD3⁺Foxp3⁺ Treg cells by flow cytometry. The results were combined from three mice.

Figure 5. Mast cell-induced IL-17 enhances suppressor function of Treg cells via upregulating CD39 and CD73.

(A) The interference of IL-17 impaired the effect of mast cells on the suppressive function of Treg cells. 5×10⁶ BMMCs were injected into tumor-bearing mice by i.v. injection. IL-17 or CCL2 neutralizing antibody or Gr-1 depleting antibody was i.p. injected to the mice 1 h, 2 days and 5 days after BMMCs injection. On day 7, tumor-infiltrating Treg cells were isolated for suppression assay. (B and C) Mast cells had no effect on Treg cells expressing CTLA-4, IL-10 or TGF-β. BMMCs were injected into tumor-bearing mice. Seven days later, the tumor-infiltrating lymphocytes were isolated for the analysis of CTLA-4 by flow cytometry. The data showed the gated CD3⁺Foxp3⁺ cells. (B) or IL-10 and TGF-β by real time RT-PCR (C). (D) Mast cells upregulated the expressions of CD39 and CD73 by Treg cells. BMMCs were injected into tumor-bearing mice with IL-17 antibody or control antibody. Seven days later, the tumor-infiltrating Treg cells were isolated for the analysis of CD39 and CD73 by real time RT-PCR and western blot. (E) IL-17 had no direct effect on Treg cells. IL-17 (20 ng/ml) was added to the cultured Treg cells for 12 hours. The cells were collected for the analysis of CD39 and CD73 by real time RT-PCR. F, Blockade of adenosine signaling pathway impaired mast cell-enhanced Treg cell function. BMMCs were injected into tumor-bearing mice. BM cells were used as control. Seven days later, the tumor-infiltrating Treg cells were isolated for suppression assay in the presence or absence of adenosine receptor A_2A antagonist SCH-58261 (100 ng/ml). The data shown were the representative of 2 independent experiments in which the similar results were obtained.

Figure 6. IL-9 strengthens the survival and protumor effect of mast cells in tumor microenvironment.

(A and B) Mast cells upregulated the expressions of IL-9 by Treg cells. BMMCs were injected into tumor-bearing mice with IL-17 antibody or control antibody. Seven days later, the tumor-infiltrating Treg cells were isolated for the analysis of IL-9 by real time RT-PCR (A). Or the isolated Treg cells were cultured for 48 hours. The supernatant was used for IL-9 ELISA assay (B). (C and D) The interference of IL-9 signaling affected mast cell-mediated MDSC infiltration and mast cell-promoted tumor growth. 5×10⁶ BMMCs were injected into tumor-bearing mice (n = 6) by i.v. injection. IL-9 neutralizing antibody or CD25 depleting antibody was i.p. injected to the mice 1 h, 2 days and 5 days after BMMCs injection. On day 12, the tumor-infiltrating lymphocytes were used to analyze Gr-1⁺CD11b⁺ MDSCs by flow cytometry (C), and the tumor growth was monitored by measuring the length (L) and width (W) of tumors. The volume (V) of the tumor was calculated by the formula V = (L×W2)/2 (D). (E) IL-9 affected the survival of mast cells in tumor microenvironment. 1×10⁶ CFSE-labeled BMMCs were directly injected into tumor tissue with multiple injection sites. IL-9 neutralizing antibody was i.p. injected to the mice 1 h, 2 days and 5 days after BMMCs injection. The tumor tissues were surgically excised on day 7 for fluorescent analysis of frozen sections.

Figure 7. A model of the closed loop among mast cells, MDSCs and Treg cells in tumor microenvironment.

Under the guidance of SCF/c-kit signaling, mast cells migrate to and are activated in tumor microenvironment; the activated mast cells release a panel of factors, leading to CCL2 production and IL-17 upregulation in MDSCs; CCL2 signaling recruits more MDSCs, leading to more IL-17 production; IL-17 strengthens tumor inflammatory microenvironment, leading to the upregulation of IL-9, IL-10, IL-13, CCL17, CCL22, CD39 and CD73; IL-10 and IL-13 induce arginase 1 expression by MDSCs; CCL17 and CCL22 attract the migration of Treg cells; CD39 and CD73 enhance suppressor function of Treg cells; IL-9 produced by Treg cells maintains the survival of mast cells; MDSCs release active MMP9, through which soluble SCF is generated, thus further facilitating the migration and activation of mast cells.