Mechanism regulating reactive oxygen species in tumor-induced myeloid-derived suppressor cells - PubMed (original) (raw)

Mechanism regulating reactive oxygen species in tumor-induced myeloid-derived suppressor cells

Cesar A Corzo et al. J Immunol. 2009.

Abstract

Myeloid-derived suppressor cells (MDSC) are a major component of the immune suppressive network described in cancer and many other pathological conditions. Recent studies have demonstrated that one of the major mechanisms of MDSC-induced immune suppression is mediated by reactive oxygen species (ROS). However, the mechanism of this phenomenon remained unknown. In this study, we observed a substantial up-regulation of ROS by MDSC in all of seven different tumor models and in patients with head and neck cancer. The increased ROS production by MDSC is mediated by up-regulated activity of NADPH oxidase (NOX2). MDSC from tumor-bearing mice had significantly higher expression of NOX2 subunits, primarily p47(phox) and gp91(phox), compared with immature myeloid cells from tumor-free mice. Expression of NOX2 subunits in MDSC was controlled by the STAT3 transcription factor. In the absence of NOX2 activity, MDSC lost the ability to suppress T cell responses and quickly differentiated into mature macrophages and dendritic cells. These findings expand our fundamental understanding of the biology of MDSC and may also open new opportunities for therapeutic regulation of these cells in cancer.

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Figures

Figure 1

Figure 1. ROS level in MDSC from tumor-bearing mice

A. Spleens from naïve tumor-free and tumor-bearing mice were collected 3 weeks after tumor injection. Splenocytes were stimulated with PMA and labeled with APC-conjugated anti-Gr-1 antibody and PerCP-conjugated anti-CD11b antibody. ROS were measured in Gr-1+CD11b+ cells by labeling cells with the oxidation-sensitive dye DCFDA as described in Materials & Methods. Each group included 4 mice. Average and standard deviation of the mean fluorescence intensity (MFI) are shown. B. H2O2 production by spleen MDSC from tumor-bearing mice. Gr-1+ CD11b+ cells were isolated from spleens of naïve, CT-26 or EL-4 tumor-bearing mice and the level of H2O2 was measured as described in Materials & Methods. C. Splenocytes (106 cells) from EL-4 tumor bearing and naïve C57BL/6 mice were loaded with 2µM DCFDA and cultured at 37°C for 30 min in RPMI-1640 in the presence of either ionomycin (2µM) or LPS (1µg/mL). Cells were then washed and stained with anti- Gr-1-APC and anti-CD11b-PE-Cy7 antibodies. ROS level was measured within the population of Gr-1+CD11b+ cells. D. MDSC from spleens of CT26, EL-4, and MC38 tumor-bearing mice were evaluated at different time points after tumor injection. Splenocytes were stimulated for 30 min with PMA and the production of ROS was determined in Gr-1+CD11b+ cells using DCFDA.

Figure 2

Figure 2. Up-regulation of ROS production in MDSC from patients with head and neck cancer

Peripheral blood MNC from healthy donors and patients with HNC were labeled with a cocktail of anti-CD11b, anti-CD14 and anti-CD33-specific antibodies and stained with DCFDA to detect ROS level within the population of CD11b+CD14−CD33+ cells. A. The gating strategy to identify MDSC. CD14−CD11b+ cells were gated first followed by gating of CD33+ cells. Histograms show representative fluorescence intensities of DCFDA in CD14−CD11b+CD33+ MDSC from patients and donors before and after PMA stimulation. B. Summarized data obtained from 5 patients and 5 healthy donors. *-statistically significant difference (p<0.05)

Figure 3

Figure 3. Up-regulation of NOX2 in MDSC

A. Gr-1+ cells were isolated from spleens of naïve or CT-26 tumor-bearing mice. RNA was extracted and expression of NADPH oxidase subunits was measured in triplicates by qRT-PCR. Three experiments with the same results were performed. *-statistically significant difference (p<0.05) between control and tumor-bearing mice. B. MDSC from spleens of CT26, EL-4, and MC38 tumor-bearing mice were evaluated at different time points after tumor injection. Gr-1+CD11b+ cells were isolated on weeks 2 and 3 after injection of tumor cells and the expression of gp91phox and p47phox was measured by qRT-PCR. Each experiment was performed in triplicates and each group included 3 mice. C, D. Protein levels of gp91phox and p47phox were determined in total cell lysate (C) or membrane fractions (D) of isolated Gr-1+CD11b+ cells. Cellular fractionation was performed using a Qiagen Cell Compartment kit.

Figure 4

Figure 4. NOX2 is responsible for ROS production in MDSC and the antigen-specific immune suppression mediated by these cells

A. Production of ROS was evaluated in splenic Gr-1+CD11b+ MDSC from EL-4 tumor-bearing and gp91phox knockout mice using staining with DCFDA. Each group included 5 mice. *-statistically significant difference (p<0.05) between wild-type and gp91phox−/− tumor-bearing mice. B, C. Gr-1+ cells were isolated from naïve, wild-type, or gp91phox KO mice and were cultured with 1×105 splenocytes from OT-1 transgenic mice. IFN-γ production (B) and cell proliferation (C) were determined after stimulation with OVA-derived specific or control peptide (10 µg/ml) as described in Materials & Methods. The values obtained from cells stimulated with control peptides were subtracted from values from cells stimulated with specific peptide. Each experiment was performed in triplicates and repeated three times. Mean and standard deviation for one representative experiment is shown. *-statistically significant difference (p<0.05) from naïve MDSC. D. Gr-1+CD11b+ cells were isolated from wild-type and gp91 KO tumor-bearing mice and cultured with 20 ng/ml GM-CSF and 25% v/v TCCM for 5 days. Cell phenotype was evaluated by flow cytometry. Cumulative results from three performed experiments are shown. *-statistically significant difference (p<0.05) from wild-type MDSC.

Figure 5

Figure 5. STAT3 regulates expression of NADPH oxidase

A, B. Nuclear extracts from STAT3C-transfected ES cells were prepared and used in EMSA. SIE –conventional STAT3 specific probe, p47phox sequence derived from promoter region of p47phox. Mutant probe – mutant p47phox derived probe, SIE cold inhibition – binding to p47phox derived probe in the presence of 50-fold excess of unlabeled SIE probe. B. ChIP assay. DNA from 32D cells was precipitated with either anti-STAT3 antibody (STAT3) or control rabbit IgG (IgG). PCR was performed with primers specific for promoter regions of p47phox or β-actin genes (C). Input – PCR reaction performed with DNA isolated from nuclear extract without precipitation. C, D, E. EL-4 tumor cells were injected into wild-type (WT) or STAT3 knockout (KO) mice. Cells from the peritoneum were flushed out and collected 21 days after injection of EL-4 cells. To recruit macrophages, thioglycollate was injected i.p. 3 days prior to sacrificing animals. Peritoneal cells were stained with anti-CD11b antibody and production of ROS was analyzed (C). CD11b+ macrophages were isolated from peritoneum of wild-type (WT) or STAT3−/− (KO) mice. The expression of p47phox was assessed by real-time PCR (D) and amount of p47phox protein was determined by Western blotting (E). F. R1-ES cells were transfected with either control plasmid (R1-C) or Stat3C plasmid (R1-Stat3C) (18). Expression of gp91phox and p47phox after transfection was determined.

Figure 6

Figure 6. Effect of STAT3 inhibitor JSI-124 on ROS level in MDSC

Gr-1+ cells were isolated from spleens of 3-week MC38 tumor-bearing mice. Cells were cultured with tumor-cell-conditioned medium for 24 hours and treated with the STAT3 inhibitor, JSI-124 (1.5 µM). A. ROS level measured in Gr-1+CD11b+ cells using DCFDA staining. B. Expression of gp91phox and p47phox at different time points after treatment with JSI-124 was evaluated using qRT-PCR. C. Levels of p47phox protein in JSI-124 treated Gr-1+CD11b+ cells were analyzed by Western blotting. Hela cells were used as a positive control for phosphorylated STAT3.

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References

    1. Kusmartsev S, Gabrilovich DI. Role Of Immature Myeloid Cells in Mechanisms of Immune Evasion In Cancer. Cancer Immunol Immunother. 2006;55:237–245. - PMC - PubMed
    1. Sica A, Bronte V. Altered macrophage differentiation and immune dysfunction in tumor development. J Clin Invest. 2007;117:1155–1166. - PMC - PubMed
    1. Talmadge JE. Pathways mediating the expansion and immunosuppressive activity of myeloid-derived suppressor cells and their relevance to cancer therapy. Clin Cancer Res. 2007;13:5243–5248. - PubMed
    1. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol. 2007;25:267–296. - PMC - PubMed
    1. Almand B, Clark JI, Nikitina E, English NR, Knight SC, Carbone DP, Gabrilovich DI. Increased production of immature myeloid cells in cancer patients. A mechanism of immunosuppression in cancer. J. Immunol. 2001;166:678–689. - PubMed

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