Expression profiles and function of IL6 in polymorphonuclear myeloid-derived suppressor cells

Abstract

IL6 is an inflammatory cytokine with pleiotropic functions in both immune and nonimmune cells, and its expression level is inversely correlated with disease prognosis in patients with cancer. However, blocking IL6 alone has only yielded minimal efficacy in human cancer patients. We aimed at defining IL6 expression profiles under inflammatory conditions and cancer, and elucidating the mechanism underlying IL6 intrinsic signaling in colon carcinoma. We report here that colonic inflammation induces IL6 expression primarily in the CD11b⁺Ly6G⁺Ly6C^lo polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in colon. Although both tumor cells, T cells and myeloid cells all express IL6, PMN-MDSCs are the primary cell type that express IL6 in colon carcinoma, suggesting that IL6 up-regulation is a response to inflammation in colon epithelium and tumor microenvironment. Furthermore, we determined that IL6 activates STAT3 to up-regulate DNMT1 and DNMT3b expression in colon tumor cells, thereby revealing an epigenetic mechanism that mediates the IL6-STAT3 signaling pathway in colon carcinoma. Surprisingly, knocking out IL6 in colon tumor cells did not significantly alter tumor growth in WT mice. Conversely, IL6-sufficient colon and pancreatic tumor grow at similar rate in WT and IL6-deficient mice. However, overexpression of IL6 in colon tumor cells significantly increases tumor growth in vivo. Our findings determine that a high tumor local IL6 threshold is essential for IL6 function in colon tumor promotion and targeting the IL6-expressing PMN-MDSCs is potentially an effective approach to suppress colon tumor growth in vivo.

Keywords: Colon tumor; Colonic inflammation; DNMT; IL6; MDSCs; STAT3.

Fig. 1

Colonic inflammation induces IL6⁺ PMN-MDSCs in colon epithelium. a Top left panel: IL6 mRNA expression level was extracted from TCGA colon Cancer (COAD) data set using the Gene using USCS Xena browser. The IL6 mRNA expression level between human colon carcinoma tissues (n = 380) and normal colon tissues (n = 51) was compared. Bottom left panel: representative image of tumor-bearing colon tissues from C57BL/6 mice treated with the AOM/DSS–water cycles. Right panels: colon tissues were collected from tumor-free (n = 3) and tumor-bearing (n = 3) mice and analyzed by semiquantitative (top panel) and qPCR (bottom panel) using mouse IL6 cDNA-specific primers. β-actin was used as an internal control. Each column in the bottom panel indicates IL6 expression level from one mouse. b RNA was extracted from the colon tissues of mice treated with the 2% DSS–water cycle at the indicated time points and analyzed for IL6 mRNA levels by RT-PCR. β-actin was used as a normalization control. c Lamina propria was extracted from colon tissue of control (n = 3) and DSS-induced colitis mice (n = 3) and stained with DAPI, and CD45- and IL6-specific mAbs. Live epithelial cell (DAPI⁻CD45⁻) and leukocytes (DAPI⁻CD45⁺) were gated out for IL6⁺ cells as shown. Bottom panel shows quantification of  % IL6⁺ cells in CD45⁻ and CD45⁺ epithelial cells and leukocytes. d Lamina propria cells were extracted from colon tissues of control (n = 3) and colitis (n = 3) mice and stained with CD11b- and Gr1-specific antibodies, followed by intracellular staining with IL-6- specific antibody. The IL-6⁺ cells were then gated and analyzed for CD11b⁺Gr1⁺ cells. Shown are representative results from one of three mice. e Quantification of  % IL-6⁺ lamina propria (top) and CD11b⁺Gr1⁺ cells (bottom). f Lamina propria was extracted from colon tissue of control (n = 3) and colitis (n = 3) mice and stained with DAPI, and CD45-, CD11b-, Ly6G-, and Ly6C-specific mAbs. Live leukocytes (DAPI⁻CD45⁺) were gated out, followed by gating for CD11b⁺, and then Ly6G⁺Ly6C^lo (PMN-MDSCs) and Ly6G⁻Ly6C^hi (M-MDSCs) cells. g Quantification and comparison of  % Ly6G⁺Ly6C^lo and Ly6G⁻Ly6C^hi cells between control and colitis mice as shown in f. h Lamina propria was extracted from colon tissue of control (n = 3) and colitis (n = 3) mice and stained with DAPI, and CD45-, CD11b-, Ly6G-, Ly6C- and IL6-specific mAbs. Live leukocytes (DAPI⁻CD45⁺) were gated out for IL6⁺ cells, followed by CD11b⁺ cells, and then Ly6G⁺Ly6C^lo and Ly6G⁻Ly6C^hi. i Quantification and comparison of % IL6⁺Ly6G⁺Ly6C^lo and IL6⁺Ly6G⁻Ly6C^hi between control and colitis mice as shown in h

Fig. 2

IL6 is primarily expressed in tumor-infiltrating PMN-MDSCs in the colon carcinoma. a Murine colon carcinoma CT26 cells were orthotopically transplanted to cecal wall of mice (n = 4). The tumor nodules were collected 4 weeks later, digested in collagenase solution to prepare single cells. The single-cell mixtures were then stained with DAPI, and CD45-, CD11b, Gr1- and IL6-specific mAbs and analyzed by flow cytometry. Shown is gating strategy. b Quantification and comparison of % IL6⁺ cells between tumor cells (CD45⁻ cells) and leukocytes (CD45⁺ cells), and between tumor cells and CD11b⁺Gr1⁺ MDSCs. C. The tumor tissue single-cell mixtures were prepared as in A, stained with DAPI, and CD45-, CD4-, CD8- and IL6-specific mAbs and analyzed by flow cytometry. Shown is the gating strategy. d Quantification and comparison of % IL6⁺ cells between CD4⁺ and CD8⁺ T cells. e The tumor tissue single-cell mixtures were prepared as in A, stained with DAPI, and CD45-, CD11b-, Ly6G- and Ly6C-specific mAbs and analyzed by flow cytometry. Shown is the gating strategy. f Quantification and comparison of % IL6⁺ cells in Ly6G⁺Ly6C^lo and Ly6G⁻Ly6C^hi cells

Fig. 3

IL6 activates the STAT3-DNMT epigenetic pathway in colon carcinoma cells. a MC38 and CT26 cells were treated with recombinant IL-6 (100 ng/mL) and IL-10 (100 ng/mL), either alone or in combination for 2 h, as indicated, and analyzed for the phosphorylated STAT3 (pSTAT3) by Western blotting. β-actin and STAT3 were used as normalization controls. b MC38 and CT26 cells were treated with recombinant IL-6 (100 ng/mL) and IL-10 (100 ng/mL), either alone or in combination for 24 h, as indicated. The cells were then analyzed for DNMT1 and DNMT3b protein levels by Western blotting. β-actin was used as a normalization control. c CT26 cells were orthotopically transplanted to cecal wall of BALB/c mice (n = 4). The tumor nodules were collected 4 weeks later, digested in collagenase solution to prepare single cells. CD45⁺ cells were purified from the digested tumor cell mixtures. Total RNA was isolated from the purified CD45⁺ cells and the CD45⁺ cell-depleted CD45⁻ cells and analyzed by qPCR for IL6 and IL10 expression level. Each column represents data from one mouse. Column: mean, Bar: SD

Fig. 4

Myeloid cells secrete IL6 to regulate STAT3 activation. a Bone marrow cells were isolated from BALB/c mice and cultured (6 × 10⁵ cells/ml) in the presence of recombinant GM-CSF protein (40 ng/ml) for 3 days to induce MDSC differentiation. J774M cells (6 × 10⁵ cells/ml) were cultured for 3 days. Culture supernatants of J774M cells and BM-derived MDSCs were collected and measured for IL6 protein level by ELISA. b J774M cells were treated with LPS (1 µg/mL) or TNF-α (1 µg/mL) for 24 h. Culture supernatants were then collected and measured for IL6 protein level by ELISA. c J774M cells were transfected with the plentiCRISPR scramble and the plentiCRISPR-il6sgRNA as described in the method. Single cells were cultured (6 × 10⁵ cells/ml), stimulated with LPS for 24 h, and IL6 protein level in the culture supernatants of each single-cell clone was determined by ELISA. Shown is IL6 protein quantification of the scramble J774M cells and three IL6 KO cell clones with undetectable IL6. d The three IL6 KO J744M single-cell clones were pooled. Culture supernatants from the J774M parent cells, J744M-Scramble cells and the pooled J774M-IL6 KO cells were collected and used to culture CT26 and MC38 cells for 24 h. Cells were then analyzed for the indicated proteins by Western blotting. e MC38 and CT26 cells were cultured in BM-MDSC-conditioned medium in the absence or presence of IL6 neutralization mAb (10 μg/ml) for 24 h. Cells were then analyzed for the indicated proteins by Western. β-actin was used as a normalization control

Fig. 5

Tumor-expressed IL6 has minimal effect on tumor growth in vivo. a CT26 and MC38 cells were transduced with lentivirus containing scramble RNA or IL6-specific sgRNA in plentiCRISPR vectors, respectively, as described in the methods. Single cells were cultured in 96-well plates and measured for IL6 protein levels by ELISA. Two single-cell clones (CT26.IL6 KO1, CT26.IL6 KO2, and MC38.IL6 KO1, MC38.IL6 KO2) with low to undetectable IL6 protein were selected and pooled (CT26.IL6 KO and MC38.IL6 KO). b CT26.scramble and CT26.IL6 KO cells were injected to the right flanks of BALB/c mice (n = 5 for each cell subline). Mice were killed 3 weeks later. Shown are tumor images. The tumor size and weight were quantified and presented at the right. c MC38.scramble and MC38.IL6 KO cells were injected to the right flanks of C57BL/6 mice (n = 10 for each cell subline). Mice were killed 3 weeks later. Shown are tumor images. The tumor size and weight were quantified and presented at the right. d MC38 cells were injected to the right flanks of WT C57BL/6 mice and IL6 KO mice. Mice were killed 3 week later. Left panel shows tumor images. The tumor size and weight were quantified and presented at the right. e Pancreatic UNKC-6141 cells were orthotopically transplanted to the pancreas of WT C57BL/6 and IL6 KO mice. Mice were killed 3 weeks later. Tumor nodules were dissected from the tumor-bearing mice and shown at the left. The tumor size and weight were quantified and presented at the right

Fig. 6

Overexpression of IL6 in tumor cells promotes colon tumor growth in vivo. a CT26 and MC38 cells were transfected with vector and IL6-expressing vector, respectively, and selected to establish stable control (CT26.vector, and MC38.vector) and IL6-overexpressing (CT26.IL6, and MC38.IL6) cell lines. Total RNA was isolated from the parent and the transfected cell lines and used for qPCR analysis of IL6 mRNA levels. The IL6 mRNA level in the parent cell lines was arbitrarily set at 1. b CT26.vector and CT26.IL6 cells were injected to the right flank of BALB/c mice (n = 5 for each cell line). Mice were killed 4 weeks later. Left panel shows tumor images of one of the two independent experiments. The tumor sizes and volume from two independent experiments were quantified and presented at the right. c MC38.vector and MC38.IL6 cells were injected to the right flank of C57BL/6 mice (n = 5 for each cell line). Mice were killed 4 weeks later. Left panel shows tumor images. The tumor sizes and volume were quantified and presented at the right