Epigenetic Component p66a Modulates Myeloid-Derived Suppressor Cells by Modifying STAT3

Jiaxuan Xin, Zhiqian Zhang, Xiaomin Su, Liyang Wang, Yuan Zhang and Rongcun Yang
J Immunol April 1, 2017, 198 (7) 2712-2720; DOI: https://doi.org/10.4049/jimmunol.1601712

Abstract

STAT3 plays a critical role in myeloid-derived suppressor cell (MDSC) accumulation and activation. Most studies have probed underlying mechanisms of STAT3 activation. However, epigenetic events involved in STAT3 activation are poorly understood. In this study, we identified several epigenetic-associated proteins such as p66a (Gatad2a), a novel protein transcriptional repressor that might interact with STAT3 in functional MDSCs, by using immunoprecipitation and mass spectrometry. p66a could regulate the phosphorylation and ubiquitination of STAT3. Silencing p66a promoted not only phosphorylation but also K63 ubiquitination of STAT3 in the activated MDSCs. Interestingly, p66a expression was significantly suppressed by IL-6 both in vitro and in vivo during MDSC activation, suggesting that p66a is involved in IL-6–mediated differentiation of MDSCs. Indeed, silencing p66a could promote MDSC accumulation, differentiation, and activation. Tumors in mice injected with p66a small interfering RNA–transfected MDSCs also grew faster, whereas tumors in mice injected with p66a-transfected MDSCs were smaller as compared with the control. Thus, our data demonstrate that p66a may physically interact with STAT3 to suppress its activity through posttranslational modification, which reveals a novel regulatory mechanism controlling STAT3 activation during myeloid cell differentiation.

Introduction

Myeloid-derived suppressor cells (MDSCs) markedly expand and participate in the suppression of immune responses in inflammation and tumor microenvironment (1, 2). MDSCs represent a heterogeneous immature cell population of the myeloid lineage that is composed of immature granulocytes, monocytes, dendritic cells, and myeloid progenitor cells (3). In mice, MDSCs are identified mainly by coexpression of myeloid lineage marker Gr1 and CD11b (4), which are further divided into two subsets: granulocytic MDSCs are characterized as CD11b+Ly6G+Ly6Clo, whereas monocytic MDSCs have a CD11b+Ly6G−Ly6Chi phenotype (5–7). In cancer and autoimmune diseases, these two subsets may play different roles (5–7). However, the suppressive functions of MDSCs are associated with the generation of arginase 1 (Arg-1), inducible NO synthase (iNOS), and reactive oxygen species, which can inhibit T cell proliferation and function (8–10). These MDSCs promote tumor growth not only by suppressing immune responses and inducing tumor tolerance, but also by promoting neoplastic progression such as tumor angiogenesis, cancer stemness, and metastasis dissemination (11–14). Thus, it is necessary to understand how these MDSCs are modulated.

Studies have shown that the expansion of MDSCs depends on several tumor-associated factors such as GM-CSF, IL-6, TNF-α, and PGE2. Most of these factors trigger the JAK-STAT3 signaling pathway in MDSCs (14–20). For instance, IL-6 binds the specific membrane-bound IL-6 receptor α-chain and the common gp130 receptor chain to induce STAT3 activation through JAK phosphorylation. STAT3 is one of the most important transcription factors that have been implicated in promoting tumor inflammation, survival, and invasion and in maintaining an immunosuppressive tumor microenvironment partially via inducing MDSCs (17, 21, 22). STAT3 deficiency markedly suppresses MDSC expansion and increases T cell responses in tumor-bearing mice (23).

STAT3 activation may be affected by posttranslational modification, including phosphorylation at C-terminal Tyr705 and Ser727 (24–29). Tyr705 (Y705) phosphorylation promotes STAT3 dimerization, nuclear translocation, and transcriptional activation (30, 31). In addition to phosphorylation, ubiquitination also plays a critical role in STAT3 activation. Ectopic expression of TMF in C2C12 cells has been reported to drive the ubiquitination and proteasomal degradation of STAT3 (32). PDLIM2 inhibits Th17 cell development through ubiquitination and degradation of STAT3 (33). Recent studies have shown that epigenetic modification plays a critical role in gene expression and cellular differentiation. These epigenetic modifications could also affect the protein phosphorylation and ubiquitination. For instance, inhibition of histone methyltransferase G9a induces Akt phosphorylation in glioma cells (34). The H3K4 methyltransferase WDR82 negatively regulates cellular antiviral response by mediating TNFR-associated factor (TRAF)3 polyubiquitination in multiple cell lines (35). However, whether the phosphorylation and ubiquitination of STAT3 are modulated by epigenetic proteins is not known.

In this study, we have found that the epigenetic component p66a, a subunit of the Mi2/NuRD/histone deacetylase (HDAC) complex, suppresses phosphorylation (Y705) and K63 ubiqitination of STAT3 by directly interacting with STAT3. Silencing p66a promotes differentiation and enhances the immune suppressive function of MDSCs, and its expression is modulated by IL-6 during MDSC activation. These results provide a novel regulatory mechanism controlling STAT3 activation in myeloid cell differentiation and offer a new strategy for improving antitumor responses.

Materials and Methods

Mice and cells

C57BL/6 and BALB/c mice were purchased from the Beijing Animal Center (Beijing, China) and maintained in a specific pathogen-free and controlled environment. B6.129S6-Il-6tm1Kopf (IL-6−/−) and CD45.1 mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China). Mice with transgenic expression of the OT-II were provided by Dr. Linrong Lu. Murine melanoma B16 and colon carcinoma CT26 were obtained from the American Type Culture Collection (Manassas, VA). 1D8 ovarian carcinoma was provided by Katherine F. Roby (University of Texas, Austin, TX). MOSEC ovarian cancer cells were gifted from Richard B.S. Roden (Johns Hopkins University, Baltimore, MD). All the cells were cultured in RPMI 1640 with 10% FBS (HyClone, Logan, UT) at 37°C in a humidified 5% CO2 atmosphere. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 8023, revised 1978).

Reagents and Abs

RPMI 1640 and FBS were obtained from HyClone. Recombinant murine GM-CSF and IL-6 were purchased from PeproTech (Rocky Hill, NJ). Rabbit anti-p66a was purchased from Bethyl Laboratories (Montgomery, TX). Rabbit anti-STAT3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti–p-STAT3 (Y705) was purchased from Cell Signaling Technology (Beverly, MA). Ubiquitin polyclonal Ab was purchased from Immunoway (Newark, DE). Polyubiquitin (K48 and K63 linkage–specific) mAb was obtained from Enzo Life Sciences (Plymouth Meeting, PA). Anti–Gr1-FITC, anti–CD11b-PE, anti–Ly6G-PE, anti–Ly6C-FITC, anti–CD4-FITC, anti–CD8-PE, anti–CD45-allophycocyanin, and anti–CD45.1-Cy7 Abs were purchased from BD Biosciences (San Diego, CA). TRAF6 and Arg-1 rabbit polyclonal Ab was purchased from Santa Cruz Biotechnology.

In vitro induction of MDSCs

Bone marrow cells (BMCs) were obtained from the femurs of C57BL/6 mice and cultured in RPMI 1640 medium supplemented with 5% FBS for 4 d in medium with GM-CSF (40 ng/ml) only or GM-CSF (40 ng/ml) plus IL-6 (40 ng/ml). To prepare the tumor cell supernatant-induced CD11b+Gr1+ MDSCs in vitro, 5 × 104 CT-26, 1D8, and MOSEC tumor cells (upper chamber) were cocultured with 2 × 106 BMCs (lower chamber) in a 24-transwell plate.

In vivo experiments

Two tumor models including C57BL/6 B16 melanoma and BALB/c CT26 colon carcinoma were used to investigate the effect of p66a-modified MDSCs on tumor growth. Mice were injected with 1 × 106 B16 or CT-26 tumor cells and were randomly divided into several experimental groups (six mice per group), and then the prepared MDSCs (1 × 106) were injected into different groups via tail vein 5, 11, and 18 d after injection of tumor cells. For the preparation of p66a-modified MDSCs, BMCs obtained from C57BL/6 CD45.1 mice (for B16 model) or BALB/c mice (for CT-26) were first transfected with control small interfering RNA (siRNA), p66a siRNA, pcDNA3.1 vector, or pcDNA3.1-p66a vector by using FuGENE HD (gene transfection) (Roche, Indianapolis, IN) or HiPerFect transfection reagent (siRNA transfection) (Qiagen, Valencia, CA) according to the manufacturer’s instructions, and then cultured with GM-CSF (40 ng/ml) plus IL-6 (40 ng/ml) for 4 d. The tumor volume was measured in two dimensions by calipers every 2 d and calculated by the following formula: Width2 × Length × π/6.

Immunoprecipitation and mass spectrometry

The MDSCs induced by GM-CSF plus IL-6 were lysed in immunoprecipitation (IP) lysis buffer (Pierce, Rockford, IL) containing 10% PMSF. Protein A/G magnetic beads (Pierce) were first added into the cell lysates for preclearing. The supernatant was collected after centrifuging at 12,000 rpm and then immunoprecipitated overnight at 4°C with the anti-STAT3 or IgG Abs. Protein A/G magnetic beads were added into cell lysates and incubated for an additional 3 h. After being washed five times, lysates were denatured and resolved by SDS-PAGE gels, silver stained, and subjected to liquid chromatography–tandem mass spectrometry sequencing and data analysis.

siRNA and gene transfection

The BMCs were collected from C57BL/6 mice and cultured in six-well plate. Cells were then transfected with pCMV-SPORT6 control, pCMV-SPORT6/STAT3, pcDNA3.1, pcDNA3.1-p66a, negative control siRNA, STAT3 siRNA, and p66a siRNA by using FuGENE HD (gene transfection) or HiPerFect transfection reagent (siRNA transfection) (Qiagen) according to the manufacturer’s instructions. STAT3 siRNA and siRNA negative control were purchased from Santa Cruz Biotechnology. p66a siRNA and negative control were purchased from Riobio (Guangzhou, China). pcDNA3.1-p66a was generated by cloning p66a and conjugating into pcDNA3.1 vectors. The pCMV-SPORT6/STAT3 complete DNA sequences (cDNA clone MGC: 67973 IMAGE: 4500786) were purchased from American Type Culture Collection.

Quantitative real-time PCR

Total RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA) and reverse transcribed to cDNA with the Moloney murine leukemia virus reverse transcriptase (Tiangen, Beijing, China) according to the manufacturer’s instructions. Quantitative PCR was performed by using UltraSYBR mixture (CWBIO, Beijing, China) and Bio-Rad iQ5 multicolor real-time RT-PCR system. GAPDH was used as an endogenous control. All the reactions were performed in triplicate. The primer sequences are listed in Supplemental Table I.

Flow cytometric analyses

Cells were collected and incubated with FITC-Gr1 or PE-CD11b Abs for 15–30 min in PBS with 1% FBS. After having been washed twice, cells were resuspended in PBS and analyzed using a FACScan flow cytometer (BD Biosciences).

Western blotting

Western blotting was performed as described previously (36). Primary Abs were used at 1:2000 dilutions and secondary Abs conjugated with HRP were used at 1:2000 dilutions.

Tumor models and cell sorting

B16 melanoma cells (1 × 106) were s.c. injected in C57BL/6 wild-type or IL-6−/− mice for 28 d to establish a tumor model in vivo. To obtain bone marrow–derived high-purity Gr1+CD11b+ cells, a CD11b and Gr1 MACS MicroBeads and cell isolation kit was used (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions.

Cytokine assay

MDSCs were obtained from GM-CSF plus IL-6–treated BMCs transfected with different siRNA for 4 d. Splenocytes were obtained from OT-II mice and stimulated by 200 nM OVA peptide (OVA323–339; GenScript, Piscataway, NJ). Both the MDSCs and splenocytes were counted by cell counting chamber. Subsequently, MDSCs and splenocytes were cocultured in 96-well plates at 37°C for 48 h at a ratio (MDSCs/OT-II splenocytes) of 1:1, 1:2, 1:5, and 1:10, respectively. The production of IFN-γ was measured by ELISA (4A Biotech, Beijing, China) according to the manufacturer’s instructions.

NO production

BMCs were cultured with GM-CSF and IL-6 for 4 d after transfection with different siRNAs. Then, the supernatant of cells was collected to detect NO by using the Griess reagent system (Promega, Madison, WI). Fifty microliters of supernatant and sulfanilamide solution was separately added into the 96-well plates in triplicate and incubated at room temperature for 5–10 min. Then, 50 μl of NED solution was added into all wells. After incubation for 5–10 min, absorbance was measured immediately at 550 nm. The concentrations of each sample were determined by using the nitrite standard reference curve.

Arginase activity assay

Briefly, equal numbers of cells were lysed with 100 μl of 0.1% Triton X-100 at 4°C for 30 min. After that, the mixture was added to 100 μl of 25 mM Tris-HCl and 10 μl of 10 mM MnCl2 and heated for 10 min at 56°C. Then, the mixture was incubated with 100 μl of 0.5 M l-arginine (pH 9.7) at 37°C for 120 min. Subsequently, the reaction was terminated with 900 μl of H2SO4/H3PO4/H2O (1:3:7). Finally, 40 μl of 9% α-isonitrosopropiophenone was added and heated for 30 min at 95°C. The concentration of urea was measured by absorbance at 540 nm. Arginase activity (unit) was defined by the amount enzyme that catalyzes the formation of 1 μg of urea per minute.

Statistical analyses

A Student t test and one-way ANOVA were used to determine statistical differences. A 95% confidence interval was considered significant and was defined as p < 0.05.

Results

p66a interacts with STAT3 in MDSCs

STAT3 activation could be modulated by several signal factors. However, some epigenetic events, which could affect the activation of STAT3, are rarely reported. Thus, we aimed to explore whether it could be modulated by epigenetic modification or interact with epigenetic modification proteins. We first examined the putative proteins, which potentially interact with STAT3. STAT3 was immunoprecipitated from BMCs treated with GM-CSF plus IL-6, and then immunoprecipitates were subjected to SDS-PAGE. Four specific protein bands (17–72 kDa) were cut from the gel for IP–mass spectrometry (MS) analysis (Fig. 1A). Gene ontology analysis revealed that abundant STAT3-interacting proteins were related to gene transcription and translation (Fig. 1B). Importantly, we found six epigenetic associated factors (SAP18, CXXC1, Ikaros, Wdr82, Wdr5, and p66a) that have an important function in regulating gene expression (Fig. 1C). Among these genes, we focused on a novel protein, p66a, which was reported as a transcription repressor in the HDAC complex Mi2/NuRD/HDAC (37, 38). To find potential interaction between p66a and STAT3, we performed a protein–protein interaction network analysis. The interaction network among STAT3, p66a, and the Mi2/NuRD/HDAC complex showed that p66a and STAT3 might interact with HDAC1/2 (Fig. 1D, 1E), suggesting that p66a may potentially interact with STAT3. To validate the interactions between p66a and STAT3, we conducted reciprocal IP followed by immunoblot. The results showed that endogenous p66a was coimmunoprecipitated with endogenous STAT3 (Fig. 1F, 1G). Meanwhile, this phenomenon was also verified in MDSCs (Gr1+CD11b+ cells) sorted from normal spleen and B16 tumor-bearing mice (Fig. 1F, 1G). Thus, these data demonstrate that p66a binds to STAT3 in bone marrow–derived MDSCs (BM-MDSCs) and natural MDSCs.

FIGURE 1.
FIGURE 1.

p66a physically interacts with STAT3 in MDSCs. (A) Identification of STAT3-interacting proteins by IP-MS. Cellular extracts from BMCs were immunoprecipitated with STAT3 Ab or isotype control and subjected to SDS-PAGE with silver staining. Arrows indicated protein bands unique to the IP products of anti-STAT3 Ab, which were excised for subsequent MS analyses. (B) Gene ontology (GO) term enrichment analyses of STAT3-interacting proteins. (C) A list of six STAT3-interacting partners associated with epigenetic modifications in BM-MDSCs identified by IP-MS. (D) Overview of the interaction network among STAT3, p66a, and the Mi2/NuRD/HDAC complex. (E) Schematic representation of the main transcription factors, signal transduction proteins, and cytokine receptors interacting with STAT3. (F and G) Validation of interactions between STAT3 and p66a in BMCs and induced MDSCs by coimmunoprecipitation assays. The data are representative of at least three independent experiments.

p66a impairs the activity of STAT3 by modulating its Y705 phosphorylation and K63 ubiquitination

Considering the relationship between STAT3 and p66a, we further investigated how p66a regulates STAT3 during MDSC activation. We collected BM-MDSCs transfected with p66a or control siRNA at different time points. Immunoblot analyses showed that silencing of p66a remarkably increased levels of phosphorylated STAT3 (Y705) in induced BM-MDSCs in a time-dependent manner (Fig. 2A–C). Meanwhile, when the immunoprecipitates of STAT3 from activated MDSCs were compared with fresh BMCs and BMCs treated with GM-CSF, we found that K63-linked ubiquitination of STAT3 was augmented in GM-CSF plus IL-6–induced MDSCs (Fig. 2D). Then, we next examined whether p66a modulates ubiquitination of STAT3 in induced MDSCs. IP assay indeed showed that silencing p66a also elevated K63-linked ubiquitination levels in the activated BM-MDSCs (Fig. 2E). Because STAT3 promotes myeloid progenitor cell proliferation through upregulation of its target genes, including Bcl-2 and c-Myc, which have STAT3 binding sites on their promoter regions (39–43), we then analyzed the mRNA level of Bcl-2 and c-Myc, and the results showed that both of them were upregulated in p66a-silenced MDSCs (Fig. 2F), indicating that the p66a may regulate STAT3-associated genes through suppressing phosphorylation and K63-linked ubiquitination of STAT3. Notably, p66a also affected K48-linked ubiquitination, suggesting that p66a also modulates the degradation of STAT3. However, our results suggest that p66a may impair the activity of MDSCs through promoting the phosphorylation and K63-linked ubiquitination of STAT3.

FIGURE 2.
FIGURE 2.

p66a inhibits phosphorylation and K63 ubiquitination of STAT3. (A) Modular structure of STAT3 and p66a. The six domains of STAT3 are N-domain, coiled-coil domain, DNA binding domain, linker domain, SH2, and transcriptional activation domain. The ubiquitination sites K48 and K63 are located at the N-terminal. Between SH2 and TAD there is a tail segment that contains the phosphorylation site Y705. For p66a, CR1 is a coiled-coil domain, and CR2 includes a GATA zinc finger domain. (B and C) Dynamic expression changes of STAT3 and p-STAT3 in p66a siRNA-transfected BM-MDSCs during GM-CSF and IL-6 treatment. Control siRNA was used as a control. (D) Pan ubiquitination and K48-linked and K63-linked ubiquitination of STAT3 in the BM-MDSCs that were induced by GM-CSF only or GM-CSF plus IL-6 for 4 d. Before immunoblot analysis, cell lysates were immunoprecipitated with STAT3 Ab. (E) Pan ubiquitination and K48-linked and K63 ubiquitiantion of STAT3 in p66a siRNA-transfected BM-MDSCs induced by GM-CSF plus IL-6. Control siRNA was used as a control. Before immunoblot analysis, cell lysates were immunoprecipitated with STAT3 Ab. (F) The mRNA expressions of STAT3 downstream target genes Bcl-2 and Myc were analyzed by quantitative RT-PCR. The data are representative of at least three independent experiments. *p < 0.05, **p < 0.01.

Previous research has shown that TRAF6 induces K63-linked ubiquitination of STAT3 (44). In our study, we have also found that TRAF6 interacts with STAT3 in BM-MDSCs, suggesting that p66a may suppress K63-linked ubiquitination of STAT3 by inhibiting the interaction between TRAF6 and STAT3.

p66a expression levels are negatively related to IL-6–mediated differentiation of MDSCs

We next determined the expression of p66a during MDSC differentiation. To realize this purpose, we established in vitro and in vivo models. In in vitro models, Gr1+CD11b+ MDSCs were successfully accumulated by 1D8 tumor cell culture supernatant or GM-CSF plus IL-6 treatment (Fig. 3A, 3B). Meanwhile, these BM-MDSCs contained significantly higher mRNA levels of iNOS, Arg-1, S100A8, and S100A9 as compared with untreated BMCs (data not shown), indicating that these cells belong to functional MDSCs. Thus, we employed microarrays to detect the levels of p66a and other related genes. Data demonstrated that BM-MDSCs had a significant reduction in mRNA levels of p66a, and also most of the other components of the Mi2/NuRD/HDAC complex compared with fresh BMCs (GEO accession no. GSE92303: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE92303; Fig. 3C). This result was validated by quantitative RT-PCR (Fig. 3D) and Western blot (Fig. 3E). However, the mRNA level of STAT3 was slightly elevated in induced BM-MDSCs compared with untreated BMCs, suggesting that p66a may not directly affect the expression of STAT3 (Fig. 3D, 3E). Importantly, the activated Gr1+CD11b+ cells sorted from BMCs and spleen of B16-bearing mice (Fig. 3F) also showed significantly lower expression of p66a than in tumor-free mice (Fig. 3G, 3H), Taken together, these data indicate the p66a levels are negatively related to the differentiation of MDSCs.

FIGURE 3.
FIGURE 3.

The expression of p66a is significantly downregulated in induced MDSCs both in vitro and in vivo. (A) Dynamic percentage changes of Gr1+CD11b+ cells induced by 1D8 tumor cell supernatant and GM-CSF plus IL-6. One representative of three independent experiments is shown. (B) Statistical analyses of the dynamic percentage changes of Gr1+CD11b+ cells in BMCs induced by 1D8 supernatant or GM-CSF plus IL-6. (C) Heat map showing the differentially expressed genes in BM-MDSCs induced by 1D8 supernatant for 4 d compared with fresh BMCs. The right panel shows the expression changes of p66a and its associated proteins in BM-MDSCs induced by 1D8 supernatant. (D) mRNA levels of STAT3 and p66a in 1D8 supernatant or GM-CSF plus IL-6–induced BM-MDSCs. Fresh BMCs were used as control. (E) p66a, STAT3, and p-STAT3 protein levels in GM-CSF plus IL-6–induced BM-MDSCs compared with fresh BMCs. (F) Percentage changes of Gr1+CD11b+ cells in BMCs and splenocytes of B16 tumor-bearing mice compared with tumor-free mice. (G and H) Detection of STAT3 and p66a expression levels in Gr1+CD11b+ MDSCs sorted from the BMCs or splenocytes of tumor-bearing mice compare with tumor-free mice (seven mice per group). The data are representative of three independent experiments. *p < 0.05, **p < 0.01.

We next addressed which cytokine could cause the reduction of p66a in our in vivo models. To accomplish this, we first observed the dynamic mRNA and protein expression levels of p66a during different factor treatment. The results showed that the reduction of p66a only appeared in BM-MDSCs under the condition of GM-CSF plus IL-6, not under GM-CSF only (Fig. 4A–C, 4E). Thus, IL-6 plays an important role in suppressing p66a expression in vivo.

FIGURE 4.
FIGURE 4.

Reduction of p66a in induced MDSCs mainly depends on the IL-6 pathway. (A and B) Dynamic mRNA expression levels of p66a in GM-CSF only (A)– or GM-CSF plus IL-6 (B)–treated BMCs. (C and D) Dynamic protein levels of p66a in GM-CSF only (C)– or GM-CSF plus IL-6 (D)–treated BMCs. (E) Quantification of p66a protein levels in GM-CSF only (A)– or GM-CSF plus IL-6 (B)–treated BMCs by ImageJ. (F) Percentage changes of Gr1+CD11b+ cells in bone marrow of B16 tumor-bearing wild-type or IL-6−/− mice compared with tumor-free mice. (G and H) mRNA (G) and protein (H) expression levels of p66a and in Gr1+CD11b+ cells sorted from the bone marrow of tumor-bearing wild-type or IL-6−/− mice, compared with tumor-free mice, respectively. The data are representative of at least three independent experiments. **p < 0.01.

To further confirm the critical role of IL-6 in regulating p66a expression during MDSC activation in vivo, we established a mouse B16 melanoma tumor model in IL-6−/− mice. B16 tumors in wild-type mice displayed larger tumor sizes than did IL-6−/− mice (Supplemental Fig. 1). The Gr1+CD11b+ cell population is increased in wild-type mice compared with those in IL-6−/− mice (Fig. 4F), supporting the conclusion that loss of IL-6 impaired MDSC accumulation in vivo. The functional Gr1+CD11b+ cells from wild-type mice had a remarkably reduced p66a as compared with those in IL-6−/− mice (Fig. 4G, 4H). Thus, these results further indicate that IL-6 contributes to the reduction of p66a during MDSC differentiation in vivo.

p66a regulates the differentiation and immune suppressive function of MDSCs

Finally, we determined the function of p66a in the differentiation and activation of MDSCs. BMCs were transfected with p66a or negative control siRNAs (Fig. 5A, 5B) and cultured with GM-CSF and IL-6 for the indicated times. Consequently, the percentage of Gr1+CD11b+ cells was increased in p66a siRNA-transfected BM-MDSCs as compared with NC siRNA-transfected BM-MDSCs in a time-dependent manner (Fig. 5C, 5E), suggesting that silencing p66a promotes the accumulation of MDSCs. Meanwhile, silencing p66a promoted the accumulation of the CD11b+LY6G−LY6Chi monocytic MDSC subset (Fig. 5D), which is consistent with the finding that STAT3 plays an important role in polarization of monocytic MDSCs (45). Furthermore, we also constructed the p66a overexpressing vector pcDNA3.1-p66a to determine the effect of p66a overexpression on the differentiation of MDSCs (Supplemental Fig. 2A, 2B). In contrast with the silence of p66a, overexpression of p66a inhibited the accumulation of MDSCs (Supplemental Fig. 2C, 2D). To detect whether the effect of p66a expression on STAT3 was specific for MDSCs, BMCs were transfected with p66a siRNA, STAT3 siRNA, or cotransfected with p66a and STAT3 siRNA. The result showed that the effect of p66a on the accumulating of MDSCs was closely associated with STAT3 (Supplemental Fig. 3). To determine the effects of p66a on the function of MDSCs, we cocultured OVA-specific OT-II CD4+ T cells with p66a siRNA-transfected BM-MDSCs. p66a siRNA-transfected BM-MDSCs showed a stronger suppression on IFN-γ secretion of OT-II T cells compared with control BM-MDSCs (Fig. 5F). We also detected the expression of functional MDSC-specific markers S100A8, S100A9, Arg-1, iNOS, and Cox2. Silencing p66a significantly increased the mRNA levels of these genes (Fig. 5G) and also promoted the production of Arg-1 and NO (Fig. 5H, 5J). These findings indicate that p66a plays a critical role in the differentiation and activation of MDSCs. We finally investigated the suppressive activity of p66a on MDSCs using in vivo experiments. MDSCs transfected with p66a siRNAs or p66a overexpressing vectors were injected into mice after tumor inoculation. As shown in Fig. 6, mice injected with p66a siRNA-transfected MDSCs presented the fastest growth (Fig. 6A, 6C), the biggest tumor size, and the heaviest tumor weight (Fig. 6B, 6D), whereas the contrary phenomena were observed in the mice injected with MDSCs transfected with pcDNA3.1-p66a, further indicating that p66a may inhibit the suppressive activity of MDSCs. Taken together, all of these suggest that p66a has a negative effect on the immune suppressive activity of MDSCs in vitro and in vivo.

FIGURE 5.
FIGURE 5.

p66a modulates the differentiation and suppressive function of MDSCs. (A and B) mRNA (A) and protein (B) levels of p66a in BMCs transfected with p66a or negative control siRNAs. (C) Dynamic percentage changes of Gr1+CD11b+ cells in induced BM-MDSCs (GM-CSF plus IL-6 treatment) transfected with p66a or control siRNAs. (D) Percentage changes of Ly6G/Ly6C MDSC subset in induced BM-MDSCs transfected with p66a or control siRNAs. (E) Statistical analyses of dynamic percentage changes of Gr1+CD11b+ cells in induced BM-MDSCs transfected with p66a or control siRNAs. (F) Suppressive capacity was performed by coculture of corresponding BM-MDSCs with the splenocytes of the OT-II transgenic mouse model with OVA323–339 stimulation. Activity of T cells was measured by their capacity to produce IFN-γ upon peptide stimulation. (G) S100A8, S100A9, Arg-1, iNOS, and Cox2 mRNA levels in induced BM-MDSCs transfected with p66a and control siRNAs. (H and I) Determination of the activities of nitrite (G) and arginase (H) in induced BM-MDSCs transfected with p66a or control siRNAs. (J) Protein levels of Arg-1 in induced BM-MDSCs transfected with p66a or control siRNAs. The data are representative of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005.

FIGURE 6.
FIGURE 6.

p66a-modified MDSCs affect tumor growth. (A and B) p66a siRNA-modified MDSCs promoted B16 melanoma growth, whereas melanoma growth could be restrained by p66a gene-modified MDSCs as compared with control. Tumor growth (A) and tumor size and tumor weight (B) in different C57BL/6 mice bearing B16 tumors (six mice per group), which were injected with MDSCs transfected with control siRNA (siNC), p66a siRNA (sip66a), pcDNA3.1 control (pcDNA3.1), and pcDNA3.1 containing the p66a gene (pcDNA3.1-p66a). (C and D) p66a siRNA-modified MDSCs promoted CT-26 colon carcinoma growth, whereas colon carcinoma growth could be restrained by p66a gene-modified MDSCs as compared with control. Tumor growth (C) and tumor size and tumor weight (D) in different BALB/c mice bearing CT-26 tumors (six mice per group), which were injected with MDSCs transfected with control siRNA (siNC), p66a siRNA (sip66a), pcDNA3.1 control (pcDNA3.1), and pcDNA3.1 containing the p66a gene (pcDNA3.1-p66a), are shown. The data are representative of three independent experiments. Days postinjection, days after injecting tumor cells. *p < 0.05, **p < 0.01.

Discussion

In this study, we demonstrate that p66a, which is suppressed by IL-6 during MDSC activation, can regulate the suppressive function and differentiation of MDSCs by interacting with STAT3. p66a not only modulates STAT3 Y705 phosphorylation but also K63-linked ubiquitination. p66a is a component of the Mi2/NuRD/HDAC histone deacetylation complex, which plays a pivotal role in the maintenance of multilineage differentiation in the early hematopoietic hierarchy (46). Our findings suggest that the Mi2/NuRD/HDAC complex may play a critical role in MDSC activation. The expansion of MDSCs depends on several critical factors such as IL-6 (14–20). IL-6 binds the specific membrane-bound IL-6 receptor α-chain and the common gp130 receptor chain to induce STAT3 activation through JAK phosphorylation. We found that p66a expression may be modulated by IL-6. Thus, our results also suggest a novel mechanism for IL-6–mediated differentiation of MDSCs. The reduced expression of p66a, which is caused by IL-6, may affect Y705 phosphorylation and K63-linked polyubiquitination of STAT3 to promote the suppressive function of MDSCs (Fig. 7).

FIGURE 7.
FIGURE 7.

Schematic representation of p66a as negative regulater of STAT3 in the IL-6 pathway. During MDSC activation, upon ligand stimulation of IL-6 receptor and other growth factor receptors, inactive STAT3 is recruited to the cytoplasmic tail of the activated receptor and subsequently activated by phosphorylation at Tyr705. STAT3 dimerizes, enters the nucleus, and activates transcription of downstream targets. In this process, p66a, which can inhibit STAT3 phosphorylation and K63-linked ubiquitination, is depressed by IL-6.

Tyrosine phosphorylation (STAT3Y705) is the most frequent phosphorylated site implicated in STAT3 activation and DNA binding. Diao et al. (16) demonstrated that exosomal Hsp70-triggered p-STAT3 contributes to MDSC suppressive activity to promote tumor growth. Our data also show that p66a physically interacts with STAT3 and suppresses its phosphorylation at Y705 site, suggesting the critical role of p66a in STAT3 activation. Meanwhile, phosphorylation of STAT3 upregulates Arg-1 in MDSCs from cancer patients (22). Our results also demonstrate that p66a silencing induces production of Arg-1, providing indirect evidence on the link of p66a and p-STAT3.

K63-linked polyubiquitination has emerged as a novel posttranslational modification for stabilizing substrates and fine-turning their functions (47). For instance, K63-linked polyubiquitination of TRAF6 has been demonstrated to create docking sites for the scaffold protein TAB2, which results in the activation of the NF-κB pathway (48). Mutation of IKKβ at Lys171 leads to a dramatic increase in K63-linked ubiquitination, which results in persistent activation of STAT3 signaling (49). However, until now, the role of K63-linked ubiquitination in STAT3 itself has been rarely reported. In the present study, we demonstrated that p66a could suppress STAT3 K63 ubiquitination. This suggests a novel mechanism for modulating the function and differentiation of MDSCS. Additionally, Wei et al. (44) demonstrated that TRAF6 mediates K63-linked ubiquitination of STAT3.

In our studies, we also found that TRAF6 interacts with STAT3 in BM-MDSCs, implying a regulatory role of TRAF6 in STAT3 ubiquitination in MDSCs.

Our data also show that p66a-modified MDSCs may affect tumor growth in vivo. Previous studies have demonstrated that MDSCs can promote the occurrence and development of multiple diseases, especially in tumors, and that STAT3 plays a critical role in the function of MDSCs and polarization of monocytic MDSCs (14, 45, 50–52). Because p66a may interact with STAT3 to affect the fucntion of MDSCs and the accumulation of MDSCs, ectopic expression of p66a can be an accurate clinical therapy for cancer patients that targets monocytic MDSCs.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Natural Science Foundation of China Grants 91029736, 9162910, and 91442111, Israel Science Foundation–National Natural Science Foundation of China Program Grant 31461143010, Ministry of Science and Technology Grant 2008AA02Z129 (Program 863), National Key Research and Development Program of China Grant 2016YFC1303604, Program for Changjiang Scholars and Innovative Research Team in University Grant IRT13023, and by funding from the State Key Laboratory of Medicinal Chemical Biology.

  • The sequences presented in this article have been submitted to the Gene Expression Omnibus under accession number GSE92303.

  • The online version of this article contains supplemental material.

  • Abbreviation used in this article:

    Arg-1
    arginase 1
    BMC
    bone marrow cell
    BM-MDSC
    bone marrow–derived myeloid-derived suppressor cell
    HDAC
    histone deacetylase
    iNOS
    inducible NO synthase
    IP
    immunoprecipitation
    MDSC
    myeloid-derived suppressor cell
    MS
    mass spectrometry
    siRNA
    small interfering RNA
    TRAF
    TNFR-associated factor.

  • Received October 5, 2016.
  • Accepted January 20, 2017.

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