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Norepinephrine-induced myeloid-derived suppressor cells block T-cell responses via generation of reactive oxygen species a
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Yufeng Liu , Jianyang Wei , Guanghui Guo & Jie Zhou a
Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China, b
Department of Microsurgery, The People's Liberation Army 371 Hospital, Xinxiang, China, and c
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Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Chinese Ministry of Education, Guangzhou, China Published online: 08 Jul 2015.
To cite this article: Yufeng Liu, Jianyang Wei, Guanghui Guo & Jie Zhou (2015) Norepinephrine-induced myeloidderived suppressor cells block T-cell responses via generation of reactive oxygen species, Immunopharmacology and Immunotoxicology, 37:4, 359-365, DOI: 10.3109/08923973.2015.1059442 To link to this article: http://dx.doi.org/10.3109/08923973.2015.1059442
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http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2015; 37(4): 359–365 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2015.1059442
RESEARCH ARTICLE
Norepinephrine-induced myeloid-derived suppressor cells block T-cell responses via generation of reactive oxygen species Yufeng Liu1*, Jianyang Wei1*, Guanghui Guo2, and Jie Zhou1,3 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China, 2Department of Microsurgery, The People’s Liberation Army 371 Hospital, Xinxiang, China, and 3Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Chinese Ministry of Education, Guangzhou, China
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Abstract
Keywords
Increased numbers of myeloid-derived suppressor cells (MDSCs) are often observed in various pathological and physiological conditions. However, the interactions between neurotransmitters and MDSCs have not been elucidated. In this study, we studied whether norepinephrine (NE), a neurotransmitter, could affect the differentiation of human MDSCs in vitro. Flow cytometric analysis showed that treatment with 20 mM NE significantly enhanced the expansion of MDSCs. The NE-generated MDSCs suppressed the T-cells proliferation, depending on the production of reactive oxygen species (ROS). Moreover, the expansion of MDSCs induced by NE resulted in a dramatic induction of nicotinamide adenine dinucleotide phosphate oxidase subunit P47phox. Addition of the ROS inhibitor catalase into the MDSCs/T-cell co-culture system partly abrogated the suppressive effects of MDSCs on T-cell proliferation. In summary, our data have shown that NE enhanced the expansion of human MDSCs in vitro, providing important insights into the novel roles of neurotransmitters in the regulation of myeloid cell differentiation and function.
Immunosuppression function, myeloid-derived suppressor cells, norepinephrine
Introduction Myeloid-derived suppressor cells (MDSCs), consisting of myeloid progenitor cells and immature myeloid cells, are abnormally accumulated in the context of several pathological and physiological conditions, including cancer, inflammatory disorders, trauma, infections and pregnancy1–3. MDSCs are defined by co-expression of the Gr1 and CD11b antigens in mice4 and generally express CD11b and the common myeloid marker CD33, while lack mature myeloid and lymphoid cell markers and the MHC class II molecule HLA-DR in humans5. There are two major subsets of MDSCs: monocytic MDSCs (M-MDSCs) and granulocytic MDSCs (G-MDSCs). MDSCs, functioning through suppression of other immune cells, are believed to be one of the key brakes in the immune system6. Depression, a major psychological disease, is a risky factor and negative prognostic indicator for many diseases, including cancer and infectious diseases7,8. Depression is known to be associated with dysregulation of the immune system. For example, T lymphocyte function is impaired in patients with depression, as shown by the shift from T-helper (Th) 2 toward Th1 cell predominance9,10. Studies of norepinephrine (NE)
*These authors contributed equally to this work. Address for correspondence: Jie Zhou, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, 74 Zhongshan, 2nd Road, Guangzhou 510080, China. Tel: +86 20 87331227. Fax: +86 20 87332588. E-mail: [email protected]
History Received 24 February 2015 Revised 28 May 2015 Accepted 31 May 2015 Published online 8 July 2015
neurotransmitter systems have revealed that inhibition of the NE transporter may represent an effective anti-depressive treatment11. Moreover, NE may regulate lymphocyte function, resulting in either protection against or progression of immune-related diseases12–14. Recent studies have shown that the neurotransmitter dopamine has anticancer activity and dramatically attenuates the inhibitory activity of tumorinduced monocytic MDSCs on T-cell proliferation and interferon (IFN)-g production15. However, the role of NE in the differentiation and function of MDSCs has not yet been elucidated. In this study, we sought to determine the regulatory effects of NE on MDSCs generation and activity. Our results provide important insights into the molecular pathways regulating the expansion of MDSCs and may facilitate the development of novel potential targeted therapies for psychological diseases, such as depression.
Materials and methods Reagents and antibodies RPMI 1640, fetal bovine serum, 5,6-carboxyfluorescein diacetate, succinimidyl ester (CFSE) and antibiotics were obtained from Invitrogen Life Technologies (Grand Island, NY). NE and oxidation-sensitive dye CM-H2DCFDA were from Sigma-Aldrich (St. Louis, MO). Human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin(IL)-6 cytokines were purchased from Peprotech
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(Oak Park, CA). The following human antibodies and their isotype controls were purchased from eBioscience (San Diego, CA): CD11b-FITC (clone, CBRM1/5), CD33-PE (clone, HIM3-4), HLA-DR-PE-Cy5 (clone, LN3), CD8a-PECy5 (clone, HIT8a), CD4-PE (clone, RPA-T4) and CD3-FITC (clone, HIT3a), CD14-Pe-Cy7 (clone, 61D3), CD15-eFlu450 (clone, HI98), CD80-FITC (clone, 2D10.4), D86-PE (clone, IT2.2), CD66b (clone, G10F5), CD163 (clone, eBioGHI/61), Human Hematopoietic Lineage APC Cocktail (22-7776-72) and corresponding isotype control antibody. Catalase, L-arginine were purchased from Sigma-Aldrich (St. Louis, MO). NW-hydroxy-nor-arginine (nor-NOHA) and L-NG-monomethyl-arginine (L -NMMA) were obtained from BioVsion (Milpitas, CA).
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Peripheral blood mononuclear cells isolation Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by whole blood Ficoll centrifugation, following the procedure described in previous reports16. PBMCs were analyzed immediately or cryopreserved at 80 C in 80% fetal calf serum, 10% RPMI 1640 and 10% DMSO. Differentiation of human MDSCs in vitro We followed the protocols described earlier16, in brief, PBMCs were cultured in a 24-well plate at 1 106 cells/ml in complete medium supplemented with GM-CSF (20 ng/ml) and IL-6 (20 ng/ml) for six days. Different concentrations of NE or PBS were added from the initiation of culture. Cultures medium were refreshed every 2–3 days. The percentages of MDSCs were evaluated by flow cytometric analysis, based on the specific surface markers HLA-DR, CD11b and CD33. Flow cytometric analysis and sorting Single-cell suspensions were prepared and stained with fluorochrome-conjugated antibodies. Data were collected on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). Data were acquired as the fraction of labeled cells within a livecell gate set for 50 000 events. For flow cytometric sorting, cells were stained with specific antibodies and isolated on a BD FACS Aria cell sorter (BD Biosciences). The purity after sorting was greater than 97%. T-cell proliferation assay T-cell proliferation assay was performed as described earlier16. CD3+T-cells were isolated from PBMCs by flow cytometric sorting, then were labeled with CFSE (3 mM) and stimulated with anti-CD3/CD28 antibodies (5 mg/ml). T-cells were cultured alone or co-cultured with autologous MDSCs at different ratios (1:0, 2:1, 4:1 and 8:1) for three days; Unstimulated T-cells were used as a negative control. Cells were harvested and stained with CD4 and CD8 antibodies. T-cell proliferation was evaluated by flow cytometric analysis, based on CFSE labeling. Reactive oxygen species (ROS) production Oxidation-sensitive dye DCFDA (dichlorodihydrofluorescein diacetate) was used to measure ROS production by MDSCs.
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Cells were labeled with HLA-DR and CD33 antibodies on 4 C for 30 min. Cells were washed with PBS. Then, it was incubated in RPMI1640 in the presence of 1 mmol DCFDA at 37 C for 15 min, and the ROS content in MDSCs was analyzed by flow cytometric. The percentage content of ROS was gated on CD33+HLA-DR MDSCs. Arginase activity assay Activity of arginase was measured in cell lysates. Briefly, 0.5 million cells were lysed with 100 ml buffer containing 0.1% Triton X-100 and 10 mM Tris-HCl for 30 min. The enzyme was activated by heating for 10 min at 56 C. Arginine hydrolysis was performed by incubating the lysate with 0.5 M L -arginine at 37 C for 120 min. Urea concentration was measured at 540 nm after the addition of a-isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 95 C for 30 min. A 1 mM urea standard and dH2O were used as controls. The arginase activity was calculated following the manufacture’s instruction (BioAssay System, Hayward, CA). Nitric oxide (NO) production NO content in supernatant was measured following the manufacturer’s protocol (Biovision, Milpitas, CA). An equal volume of plasma (100 ml) was mixed with the Greiss reagent and incubated for 10 min at room temperature. The absorbance at 550 nm was measured using microplate reader (Bio-Rad, Hercules, CA). Nitrite concentrations were determined by comparing the absorbance values for the test samples to a standard curve generated by serial dilution of 0.25 mM sodium nitrite. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) RNA was extracted using an RNase Mini kit and reverse transcribed into cDNA using SuperScript III Reverse Transcriptase kit (QIAGEN, Valencia, CA). qPCR was performed as described briefly: qPCR amplification was performed using SYBR Green (Takara, Otsu, Japan) with gene-specific primers (Table 1) in Bio-rad thermal cycler. A single 10 ml mixture of qRT-PCR was prepared to each PCR optical tube, with 5 ml of 2 SYBR Premix Ex Taq, 1 ml qPCR forward primer (10 mM) and reverse primer (10 mM), 1 ml of diluted cDNA (5 ng/ml) and 3 ml autoclaved deionized water. After reaction, the specificity of qRT-PCR products and amplification efficiency were determined by the melting curves and amplification curves, respectively. The relative expression levels of specific genes were calculated based on Table 1. Sequences of primers used in this study. Primer sequence Arg1-for Arg1-rev NOS2-for NOS2-rev P47phox-for P47phox-rev b-Actin-for b-Actin-rev
50 -ATTATCGGAGCGCCTTTCTC-30 50 -ACAGACCGTGGGTTCTTCAC-30 50 -TTCAGTATCACAACCTCAGCAAG-30 50 -TGGACCTGCAAGTTAAAATCCC-30 50 -CCACACCTGCTGGACTTCTT-30 50 -GCCACGGTCATCTCTGTTC-30 50 -GCTCTTTTCCAGCCTTCCTT-30 50 -CATACAGGTCTTTGCGGATGT-30
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the cycle number of the peaks (Ct value) and normalized to internal control b-actin. The reactions were performed in triplicate, and the data were analyzed by SPSS software (SPSS Inc., Chicago, IL). Statistics The data are presented as mean ± standard deviation (SD). Data analyze was done with t-tests between two groups and ANOVA followed by Bonferroni or Dunnett t-test for multiple comparisons. Statistical tests were performed using Graph Pad Prism 5.0 (Graph Pad Software Inc., La Jolla, CA), and SPSS, version 18.0 software (SPSS Inc., Chicago, IL). *p50.05, **p50.01 were considered statistically significant.
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Results NE enhanced the differentiation of M-MDSCs in vitro To determine whether administration of NE affected the differentiation of human MDSCs, in vitro culture of PBMCs was performed under previously described conditions16. Different concentrations of NE were added at the initiation of culture, and the percentages of MDSCs (HLA-DR/ low CD11b+CD33+ cells) were evaluated by flow cytometry. We found that administration of 20 mM NE significantly increased the population of MDSCs compared with the control treatment (Figure 1A and B). To further determine the subset of MDSCs generated by NE, we evaluated the expression of CD14 and CD15 molecules. Results showed that NE-induced MDSCs were CD14 positive, but lack the expression of CD15, supporting that they were M-MDSCs (HLA-DR/ low CD11b+CD33+CD14+) subset (Figure 1C). To further determine the phenotype of M-MDSCs, we compared the
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expression of others molecules phenotypic markers including CD80, CD86, etc. Representative histograms of the expression of surface molecules in M-MDSCs are shown in Supplemental Figure 1. As reported, human M-MDSCs expressed CD14 positive and Lin negative17. We found that NE-induced M-MDSCs expressed high level of CD14, but not CD15 and Lin (that is CD3, CD19, CD56 and CD13) expression. In conclusion, we determined that NE induced M-MDSCs expansion in vitro. NE-generated MDSCs suppressed T-cell proliferation MDSCs are known to suppress immune responses through a variety of mechanisms depending on the specific pathological or physiology conditions. As reported, GM-CSF- and IL6-induced MDSCs could suppress T-cell function18. In our study, cytokine-induced MDSCs were purified by flow cytometry and co-cultured with stimulated autologous T-cells at different ratios for 3 days. T cell proliferation was then measured by CFSE dilution. Consistent with previous reporter, the MDSCs generated by GM-CSF and IL-6 (control) displayed suppressive activity (Figure 2A). Therefore, we next determined whether NE-induced MDSCs was much stronger than cytokines-induced MDSCs in suppressing T-cell proliferation. Cytokine and NE-induced MDSCs were purified by flow cytometry and used for T-cell proliferation assay. We found that NE-induced MDSCs significantly suppressed the proliferation of CD4+ and CD8+ T-cells (Figure 2A and B) and blocked IFN-g production (Figure 2C) when the ratio of MDSCs to T-cells was 1:4 or 1:2. Furthermore, suppress capability of NE-derived MDSCs was much stronger than control in suppressing T-cell proliferation (Figure 2A and B). In addition to determined that MDSCs suppress autologous
Figure 1. NE enhanced the differentiation of MDSCs in vitro. PBMCs from healthy donors were cultured with GM-CSF (20 ng/mL) and IL-6 (20 ng/ mL) for 6 days. Different concentrations of NE were added at the beginning of culture. PBS was used as control. The expression of surface markers for human MDSCs was analyzed by flow cytometry, and the proportion of MDSCs (HLA-DR/lowCD11b+CD33+) is presented. (A) Representative flow cytometric data from a single experiment. (B) Results are the mean ± SD from healthy individuals (n ¼ 6) and measurements were repeated three times. (C) Expression of CD14 and CD15 was analyzed by flow cytometry to determine the MDSCs subset; *p50.05, **p50.01 compared with the control group.
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Figure 2. NE-generated MDSCs suppressed T-cell proliferation. Control and NE-induced MDSCs were purified by flow cytometry and co-cultured with stimulated autologous T cells at different ratios for 3 days. T-cell proliferation was measured by CFSE dilution. T cells without stimulation were used as a negative control. (A) Representative data from a single experiment. (B) Statistical analysis of the data from four donors (n ¼ 4) and measurements were repeated three times for group. (C) Production of IFN-g by T cells was measured in supernatants from (A) using ELISA. Each group included four donors and measurements were repeated three times. *p50.05, **p50.01 compared with the control group.
T-cells proliferation, we further detected whether co-cultured with allogeneic T-cell could also suppress T-cells proliferation. As shown in Supplemental Figure 2, we found that NE-generated MDSCs displayed corresponding suppressive activity when co-culture with allogeneic-derived T-cell. Changes in L-arginine metabolism in MDSCs induced by NE As our data showed that NE-generated MDSCs suppressed the T-cell response, we further investigated the mechanism of MDSC-mediated T-cell suppression. MDSCs utilize a number of mechanisms to suppress T-cell function, including high levels of arginase activity, nitric oxide (NO) production and ROS generation18. We therefore compared the levels of arginase, NO and ROS in NE-generated and control MDSCs. We found that ROS production in NE-generated MDSCs (HLA-DRCD33+) was increased dramatically compared to control MDSCs (Figure 3A), while there were no changes in the levels of arginase activity (Figure 3B) and NO content (Figure 3C). We next examined the expression of enzymes responsible for L-arginine metabolism by qRT-PCR in purified MDSCs from NE and control-generated MDSCs.
No changes were observed in the expression levels of the Arg1 and NOS2 genes, which are responsible for arginase activity and NO production, respectively (Figure 3D). We found that the expression of p47phox was increased in NE-generated MDSCs. Supporting the essential role of ROS in immune suppression mediated by NE-generated MDSCs. This effect was further confirmed by analysis of protein expression by Western blotting (Figure 3E). The suppressive activity of NE-induced MDSCs was dependent on ROS Addition of the ROS inhibitor (Catalase) into the MDSC/Tcell co-culture system partially abrogated the suppressive effects of MDSCs on T-cell proliferation (Figure 4A) and IFN-g production (Figure 4B), supporting the role of ROS in immune suppression mediated by NE-induced MDSCs. In contrast, inhibitors for arginase or iNOS, or L-arginine supplementation had no effect.
Discussion MDSCs are known to suppress immune function and promote the progression of several diseases19,20. However, the
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Figure 3. Changes in L-arginine metabolism in MDSCs induced by NE. (A) ROS production in MDSCs was measured by flow cytometry. HLA-DR/ low CD33+ cells were gated, and the percentage of CM-H2DCFDA+ cells is shown as the mean ± SEM of six samples. (B) Arginase activity in MDSCs and (C) no content in supernatants was detected as previously described. (D) The expression levels of Arg1, NOS2 and p47phox in MDSCs were measured by qRT-PCR. (E) The expression levels of Arg1, NOS2 and p47phox in MDSCs were measured by WB. Each group included six donors and measurements were repeated three times for each group. *p50.05, **p50.01 compared with the control group.
Figure 4. The suppressive activity of NE-induced MDSCs was dependent on ROS. (A) The effects of different inhibitors on the function of NEgenerated MDSCs were evaluated using the allogeneic mixed lymphocyte reaction. T cells were stimulated with anti-CD3/CD28 antibodies, cocultured with MDSCs from the same donors at a 2:1 ratio with the indicated treatments for 3 days and evaluated for T-cell proliferation by CFSE dilution. Unstimulated T cells were used as a negative control. nor-NOHA, arginase inhibitor; L-NMMA, iNOS inhibitor; catalase, ROS inhibitor; LNMMA, NOS inhibitor; L-arginine. T cells alone were used as a positive control. (B) Statistical analysis of the effects of the inhibitors on the suppressive effects of MDSCs. (C) Production of IFN-g by T cells in supernatants from (A) as measured by ELISA. Each group included six donors and measurements were repeated three times for each group. *p50.05, **p50.01 compared with the control group.
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interactions between MDSCs and neurotransmitters have not been elucidated. Here, we showed that NE, the primary neurotransmitter for the sympathetic nervous system, promoted the expansion of MDSCs and enhanced the immunosuppressive activities of MDSCs. Therefore, our results suggested that MDSCs may represent a novel target for pharmacologic intervention in psychological diseases. NE has been reported to have broad immunomodulatory effects. Reports shown that NE modulates cellular activity by binding to adrenergic receptors expressed on various types of cells, such as T and B cells21,22. Moreover, NE can regulate immune cell function during infection and immune challenge23. In this study, we found that NE could enhance the differentiation of human MDSCs in vitro. Additionally, NE-generated MDSCs exhibited immunosuppressive functions much stronger than control MDSCs, and this effect of NE was dependent on ROS production. We also showed that the enhanced ROS production in NE-generated MDSCs was associated with the induction of the NADPH subunit p47phox. This result was consistent with a previous report, which showed that NE increases NADPH oxidase-derived superoxide in human PBMCs24. Given that the studies have reported that MDSCs are believed to be one of the key brakes of immune system function. In our study, our data shown that NE enhanced the differentiation of human MDSCs in vitro, providing important insights into the novel roles of neurotransmitters in the regulation of myeloid cell differentiation and function. Anti-depressant therapy usually inhibits neurotransmitter release, just as NE. We hypothesized that anti-depressant drugs inhibit the release of NE, thereby reduce the expansion of MDSCs, eventually lead to the recovery of the body’s immune response. Therefore, we speculate that MDSCs may represent the potential targets and prognostic indicators of anti-depressant treatment. Of course, the next step we need to analysis is abundant clinical samples to confirm this hypothesis. In our study, we used PBMCs as our cellular model since these cells mimic the physiological hematopoietic environment in circulation; this allowed us to analyze crosstalk between neurotransmitters and immune cells25. Thus, functionally, the expansion of MDSCs induced by NE, as observed from these heterogeneous cell populations, was considered to be more representative of an intact system than analysis of individual populations of cells. Furthermore, it is important to note that these mutual effects between neurotransmitters and immune cells were observed in primary human cells and not from an immortalized cell line, providing a foundation for future patient studies to examine cellular mechanisms and potentially elucidate biomarkers for health and disease26. However, it is still unclear whether NE induces the expansion and functional activation of MDSCs in vivo; thus, additional studies are required to investigate this topic. In summary, our study showed that NE induced the expansion and activation of MDSCs, suppressing immune system function. These data provide important insights into the immunomodulatory effects of NE. Meanwhile, our findings may also raise concerns regarding the potential immunoregulatory effects of NE by facilitating the differentiation and activation of MDSCs.
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Declaration of interest The authors have no conflicts of interest to declare. This work was supported by the following grants to J.Z: National Key Basic Research Program of China (No. 2012CB524900), Guangdong Innovative Research Team Program (No. 2009010058), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (GDUPS, 2014), Program for the 12th Five-year Plan (No. 2012ZX10001003), National Natural Science Foundation of China (No. 81072397; No. 31270921), Natural Science Foundation of Guangdong (No. S2011020006072), the Fundamental Research Funds for the Central Universities, the Provincial Talents Cultivated by ‘‘Thousand-HundredTen’’ program of Guangdong Province, 111 Project (No. B12003).
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Supplementary materials are available in on-line Supplemental Figures 1 and 2
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Norepinephrine-induced myeloid-derived suppressor cells block T-cell responses via generation of reactive oxygen species a
a
b
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Yufeng Liu , Jianyang Wei , Guanghui Guo & Jie Zhou a
Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China, b
Department of Microsurgery, The People's Liberation Army 371 Hospital, Xinxiang, China, and c
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Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Chinese Ministry of Education, Guangzhou, China Published online: 08 Jul 2015.
To cite this article: Yufeng Liu, Jianyang Wei, Guanghui Guo & Jie Zhou (2015) Norepinephrine-induced myeloidderived suppressor cells block T-cell responses via generation of reactive oxygen species, Immunopharmacology and Immunotoxicology, 37:4, 359-365, DOI: 10.3109/08923973.2015.1059442 To link to this article: http://dx.doi.org/10.3109/08923973.2015.1059442
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http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, 2015; 37(4): 359–365 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2015.1059442
RESEARCH ARTICLE
Norepinephrine-induced myeloid-derived suppressor cells block T-cell responses via generation of reactive oxygen species Yufeng Liu1*, Jianyang Wei1*, Guanghui Guo2, and Jie Zhou1,3 Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China, 2Department of Microsurgery, The People’s Liberation Army 371 Hospital, Xinxiang, China, and 3Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Chinese Ministry of Education, Guangzhou, China
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Abstract
Keywords
Increased numbers of myeloid-derived suppressor cells (MDSCs) are often observed in various pathological and physiological conditions. However, the interactions between neurotransmitters and MDSCs have not been elucidated. In this study, we studied whether norepinephrine (NE), a neurotransmitter, could affect the differentiation of human MDSCs in vitro. Flow cytometric analysis showed that treatment with 20 mM NE significantly enhanced the expansion of MDSCs. The NE-generated MDSCs suppressed the T-cells proliferation, depending on the production of reactive oxygen species (ROS). Moreover, the expansion of MDSCs induced by NE resulted in a dramatic induction of nicotinamide adenine dinucleotide phosphate oxidase subunit P47phox. Addition of the ROS inhibitor catalase into the MDSCs/T-cell co-culture system partly abrogated the suppressive effects of MDSCs on T-cell proliferation. In summary, our data have shown that NE enhanced the expansion of human MDSCs in vitro, providing important insights into the novel roles of neurotransmitters in the regulation of myeloid cell differentiation and function.
Immunosuppression function, myeloid-derived suppressor cells, norepinephrine
Introduction Myeloid-derived suppressor cells (MDSCs), consisting of myeloid progenitor cells and immature myeloid cells, are abnormally accumulated in the context of several pathological and physiological conditions, including cancer, inflammatory disorders, trauma, infections and pregnancy1–3. MDSCs are defined by co-expression of the Gr1 and CD11b antigens in mice4 and generally express CD11b and the common myeloid marker CD33, while lack mature myeloid and lymphoid cell markers and the MHC class II molecule HLA-DR in humans5. There are two major subsets of MDSCs: monocytic MDSCs (M-MDSCs) and granulocytic MDSCs (G-MDSCs). MDSCs, functioning through suppression of other immune cells, are believed to be one of the key brakes in the immune system6. Depression, a major psychological disease, is a risky factor and negative prognostic indicator for many diseases, including cancer and infectious diseases7,8. Depression is known to be associated with dysregulation of the immune system. For example, T lymphocyte function is impaired in patients with depression, as shown by the shift from T-helper (Th) 2 toward Th1 cell predominance9,10. Studies of norepinephrine (NE)
*These authors contributed equally to this work. Address for correspondence: Jie Zhou, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, 74 Zhongshan, 2nd Road, Guangzhou 510080, China. Tel: +86 20 87331227. Fax: +86 20 87332588. E-mail: [email protected]
History Received 24 February 2015 Revised 28 May 2015 Accepted 31 May 2015 Published online 8 July 2015
neurotransmitter systems have revealed that inhibition of the NE transporter may represent an effective anti-depressive treatment11. Moreover, NE may regulate lymphocyte function, resulting in either protection against or progression of immune-related diseases12–14. Recent studies have shown that the neurotransmitter dopamine has anticancer activity and dramatically attenuates the inhibitory activity of tumorinduced monocytic MDSCs on T-cell proliferation and interferon (IFN)-g production15. However, the role of NE in the differentiation and function of MDSCs has not yet been elucidated. In this study, we sought to determine the regulatory effects of NE on MDSCs generation and activity. Our results provide important insights into the molecular pathways regulating the expansion of MDSCs and may facilitate the development of novel potential targeted therapies for psychological diseases, such as depression.
Materials and methods Reagents and antibodies RPMI 1640, fetal bovine serum, 5,6-carboxyfluorescein diacetate, succinimidyl ester (CFSE) and antibiotics were obtained from Invitrogen Life Technologies (Grand Island, NY). NE and oxidation-sensitive dye CM-H2DCFDA were from Sigma-Aldrich (St. Louis, MO). Human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin(IL)-6 cytokines were purchased from Peprotech
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(Oak Park, CA). The following human antibodies and their isotype controls were purchased from eBioscience (San Diego, CA): CD11b-FITC (clone, CBRM1/5), CD33-PE (clone, HIM3-4), HLA-DR-PE-Cy5 (clone, LN3), CD8a-PECy5 (clone, HIT8a), CD4-PE (clone, RPA-T4) and CD3-FITC (clone, HIT3a), CD14-Pe-Cy7 (clone, 61D3), CD15-eFlu450 (clone, HI98), CD80-FITC (clone, 2D10.4), D86-PE (clone, IT2.2), CD66b (clone, G10F5), CD163 (clone, eBioGHI/61), Human Hematopoietic Lineage APC Cocktail (22-7776-72) and corresponding isotype control antibody. Catalase, L-arginine were purchased from Sigma-Aldrich (St. Louis, MO). NW-hydroxy-nor-arginine (nor-NOHA) and L-NG-monomethyl-arginine (L -NMMA) were obtained from BioVsion (Milpitas, CA).
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Peripheral blood mononuclear cells isolation Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by whole blood Ficoll centrifugation, following the procedure described in previous reports16. PBMCs were analyzed immediately or cryopreserved at 80 C in 80% fetal calf serum, 10% RPMI 1640 and 10% DMSO. Differentiation of human MDSCs in vitro We followed the protocols described earlier16, in brief, PBMCs were cultured in a 24-well plate at 1 106 cells/ml in complete medium supplemented with GM-CSF (20 ng/ml) and IL-6 (20 ng/ml) for six days. Different concentrations of NE or PBS were added from the initiation of culture. Cultures medium were refreshed every 2–3 days. The percentages of MDSCs were evaluated by flow cytometric analysis, based on the specific surface markers HLA-DR, CD11b and CD33. Flow cytometric analysis and sorting Single-cell suspensions were prepared and stained with fluorochrome-conjugated antibodies. Data were collected on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). Data were acquired as the fraction of labeled cells within a livecell gate set for 50 000 events. For flow cytometric sorting, cells were stained with specific antibodies and isolated on a BD FACS Aria cell sorter (BD Biosciences). The purity after sorting was greater than 97%. T-cell proliferation assay T-cell proliferation assay was performed as described earlier16. CD3+T-cells were isolated from PBMCs by flow cytometric sorting, then were labeled with CFSE (3 mM) and stimulated with anti-CD3/CD28 antibodies (5 mg/ml). T-cells were cultured alone or co-cultured with autologous MDSCs at different ratios (1:0, 2:1, 4:1 and 8:1) for three days; Unstimulated T-cells were used as a negative control. Cells were harvested and stained with CD4 and CD8 antibodies. T-cell proliferation was evaluated by flow cytometric analysis, based on CFSE labeling. Reactive oxygen species (ROS) production Oxidation-sensitive dye DCFDA (dichlorodihydrofluorescein diacetate) was used to measure ROS production by MDSCs.
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Cells were labeled with HLA-DR and CD33 antibodies on 4 C for 30 min. Cells were washed with PBS. Then, it was incubated in RPMI1640 in the presence of 1 mmol DCFDA at 37 C for 15 min, and the ROS content in MDSCs was analyzed by flow cytometric. The percentage content of ROS was gated on CD33+HLA-DR MDSCs. Arginase activity assay Activity of arginase was measured in cell lysates. Briefly, 0.5 million cells were lysed with 100 ml buffer containing 0.1% Triton X-100 and 10 mM Tris-HCl for 30 min. The enzyme was activated by heating for 10 min at 56 C. Arginine hydrolysis was performed by incubating the lysate with 0.5 M L -arginine at 37 C for 120 min. Urea concentration was measured at 540 nm after the addition of a-isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 95 C for 30 min. A 1 mM urea standard and dH2O were used as controls. The arginase activity was calculated following the manufacture’s instruction (BioAssay System, Hayward, CA). Nitric oxide (NO) production NO content in supernatant was measured following the manufacturer’s protocol (Biovision, Milpitas, CA). An equal volume of plasma (100 ml) was mixed with the Greiss reagent and incubated for 10 min at room temperature. The absorbance at 550 nm was measured using microplate reader (Bio-Rad, Hercules, CA). Nitrite concentrations were determined by comparing the absorbance values for the test samples to a standard curve generated by serial dilution of 0.25 mM sodium nitrite. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) RNA was extracted using an RNase Mini kit and reverse transcribed into cDNA using SuperScript III Reverse Transcriptase kit (QIAGEN, Valencia, CA). qPCR was performed as described briefly: qPCR amplification was performed using SYBR Green (Takara, Otsu, Japan) with gene-specific primers (Table 1) in Bio-rad thermal cycler. A single 10 ml mixture of qRT-PCR was prepared to each PCR optical tube, with 5 ml of 2 SYBR Premix Ex Taq, 1 ml qPCR forward primer (10 mM) and reverse primer (10 mM), 1 ml of diluted cDNA (5 ng/ml) and 3 ml autoclaved deionized water. After reaction, the specificity of qRT-PCR products and amplification efficiency were determined by the melting curves and amplification curves, respectively. The relative expression levels of specific genes were calculated based on Table 1. Sequences of primers used in this study. Primer sequence Arg1-for Arg1-rev NOS2-for NOS2-rev P47phox-for P47phox-rev b-Actin-for b-Actin-rev
50 -ATTATCGGAGCGCCTTTCTC-30 50 -ACAGACCGTGGGTTCTTCAC-30 50 -TTCAGTATCACAACCTCAGCAAG-30 50 -TGGACCTGCAAGTTAAAATCCC-30 50 -CCACACCTGCTGGACTTCTT-30 50 -GCCACGGTCATCTCTGTTC-30 50 -GCTCTTTTCCAGCCTTCCTT-30 50 -CATACAGGTCTTTGCGGATGT-30
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the cycle number of the peaks (Ct value) and normalized to internal control b-actin. The reactions were performed in triplicate, and the data were analyzed by SPSS software (SPSS Inc., Chicago, IL). Statistics The data are presented as mean ± standard deviation (SD). Data analyze was done with t-tests between two groups and ANOVA followed by Bonferroni or Dunnett t-test for multiple comparisons. Statistical tests were performed using Graph Pad Prism 5.0 (Graph Pad Software Inc., La Jolla, CA), and SPSS, version 18.0 software (SPSS Inc., Chicago, IL). *p50.05, **p50.01 were considered statistically significant.
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Results NE enhanced the differentiation of M-MDSCs in vitro To determine whether administration of NE affected the differentiation of human MDSCs, in vitro culture of PBMCs was performed under previously described conditions16. Different concentrations of NE were added at the initiation of culture, and the percentages of MDSCs (HLA-DR/ low CD11b+CD33+ cells) were evaluated by flow cytometry. We found that administration of 20 mM NE significantly increased the population of MDSCs compared with the control treatment (Figure 1A and B). To further determine the subset of MDSCs generated by NE, we evaluated the expression of CD14 and CD15 molecules. Results showed that NE-induced MDSCs were CD14 positive, but lack the expression of CD15, supporting that they were M-MDSCs (HLA-DR/ low CD11b+CD33+CD14+) subset (Figure 1C). To further determine the phenotype of M-MDSCs, we compared the
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expression of others molecules phenotypic markers including CD80, CD86, etc. Representative histograms of the expression of surface molecules in M-MDSCs are shown in Supplemental Figure 1. As reported, human M-MDSCs expressed CD14 positive and Lin negative17. We found that NE-induced M-MDSCs expressed high level of CD14, but not CD15 and Lin (that is CD3, CD19, CD56 and CD13) expression. In conclusion, we determined that NE induced M-MDSCs expansion in vitro. NE-generated MDSCs suppressed T-cell proliferation MDSCs are known to suppress immune responses through a variety of mechanisms depending on the specific pathological or physiology conditions. As reported, GM-CSF- and IL6-induced MDSCs could suppress T-cell function18. In our study, cytokine-induced MDSCs were purified by flow cytometry and co-cultured with stimulated autologous T-cells at different ratios for 3 days. T cell proliferation was then measured by CFSE dilution. Consistent with previous reporter, the MDSCs generated by GM-CSF and IL-6 (control) displayed suppressive activity (Figure 2A). Therefore, we next determined whether NE-induced MDSCs was much stronger than cytokines-induced MDSCs in suppressing T-cell proliferation. Cytokine and NE-induced MDSCs were purified by flow cytometry and used for T-cell proliferation assay. We found that NE-induced MDSCs significantly suppressed the proliferation of CD4+ and CD8+ T-cells (Figure 2A and B) and blocked IFN-g production (Figure 2C) when the ratio of MDSCs to T-cells was 1:4 or 1:2. Furthermore, suppress capability of NE-derived MDSCs was much stronger than control in suppressing T-cell proliferation (Figure 2A and B). In addition to determined that MDSCs suppress autologous
Figure 1. NE enhanced the differentiation of MDSCs in vitro. PBMCs from healthy donors were cultured with GM-CSF (20 ng/mL) and IL-6 (20 ng/ mL) for 6 days. Different concentrations of NE were added at the beginning of culture. PBS was used as control. The expression of surface markers for human MDSCs was analyzed by flow cytometry, and the proportion of MDSCs (HLA-DR/lowCD11b+CD33+) is presented. (A) Representative flow cytometric data from a single experiment. (B) Results are the mean ± SD from healthy individuals (n ¼ 6) and measurements were repeated three times. (C) Expression of CD14 and CD15 was analyzed by flow cytometry to determine the MDSCs subset; *p50.05, **p50.01 compared with the control group.
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Figure 2. NE-generated MDSCs suppressed T-cell proliferation. Control and NE-induced MDSCs were purified by flow cytometry and co-cultured with stimulated autologous T cells at different ratios for 3 days. T-cell proliferation was measured by CFSE dilution. T cells without stimulation were used as a negative control. (A) Representative data from a single experiment. (B) Statistical analysis of the data from four donors (n ¼ 4) and measurements were repeated three times for group. (C) Production of IFN-g by T cells was measured in supernatants from (A) using ELISA. Each group included four donors and measurements were repeated three times. *p50.05, **p50.01 compared with the control group.
T-cells proliferation, we further detected whether co-cultured with allogeneic T-cell could also suppress T-cells proliferation. As shown in Supplemental Figure 2, we found that NE-generated MDSCs displayed corresponding suppressive activity when co-culture with allogeneic-derived T-cell. Changes in L-arginine metabolism in MDSCs induced by NE As our data showed that NE-generated MDSCs suppressed the T-cell response, we further investigated the mechanism of MDSC-mediated T-cell suppression. MDSCs utilize a number of mechanisms to suppress T-cell function, including high levels of arginase activity, nitric oxide (NO) production and ROS generation18. We therefore compared the levels of arginase, NO and ROS in NE-generated and control MDSCs. We found that ROS production in NE-generated MDSCs (HLA-DRCD33+) was increased dramatically compared to control MDSCs (Figure 3A), while there were no changes in the levels of arginase activity (Figure 3B) and NO content (Figure 3C). We next examined the expression of enzymes responsible for L-arginine metabolism by qRT-PCR in purified MDSCs from NE and control-generated MDSCs.
No changes were observed in the expression levels of the Arg1 and NOS2 genes, which are responsible for arginase activity and NO production, respectively (Figure 3D). We found that the expression of p47phox was increased in NE-generated MDSCs. Supporting the essential role of ROS in immune suppression mediated by NE-generated MDSCs. This effect was further confirmed by analysis of protein expression by Western blotting (Figure 3E). The suppressive activity of NE-induced MDSCs was dependent on ROS Addition of the ROS inhibitor (Catalase) into the MDSC/Tcell co-culture system partially abrogated the suppressive effects of MDSCs on T-cell proliferation (Figure 4A) and IFN-g production (Figure 4B), supporting the role of ROS in immune suppression mediated by NE-induced MDSCs. In contrast, inhibitors for arginase or iNOS, or L-arginine supplementation had no effect.
Discussion MDSCs are known to suppress immune function and promote the progression of several diseases19,20. However, the
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Figure 3. Changes in L-arginine metabolism in MDSCs induced by NE. (A) ROS production in MDSCs was measured by flow cytometry. HLA-DR/ low CD33+ cells were gated, and the percentage of CM-H2DCFDA+ cells is shown as the mean ± SEM of six samples. (B) Arginase activity in MDSCs and (C) no content in supernatants was detected as previously described. (D) The expression levels of Arg1, NOS2 and p47phox in MDSCs were measured by qRT-PCR. (E) The expression levels of Arg1, NOS2 and p47phox in MDSCs were measured by WB. Each group included six donors and measurements were repeated three times for each group. *p50.05, **p50.01 compared with the control group.
Figure 4. The suppressive activity of NE-induced MDSCs was dependent on ROS. (A) The effects of different inhibitors on the function of NEgenerated MDSCs were evaluated using the allogeneic mixed lymphocyte reaction. T cells were stimulated with anti-CD3/CD28 antibodies, cocultured with MDSCs from the same donors at a 2:1 ratio with the indicated treatments for 3 days and evaluated for T-cell proliferation by CFSE dilution. Unstimulated T cells were used as a negative control. nor-NOHA, arginase inhibitor; L-NMMA, iNOS inhibitor; catalase, ROS inhibitor; LNMMA, NOS inhibitor; L-arginine. T cells alone were used as a positive control. (B) Statistical analysis of the effects of the inhibitors on the suppressive effects of MDSCs. (C) Production of IFN-g by T cells in supernatants from (A) as measured by ELISA. Each group included six donors and measurements were repeated three times for each group. *p50.05, **p50.01 compared with the control group.
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interactions between MDSCs and neurotransmitters have not been elucidated. Here, we showed that NE, the primary neurotransmitter for the sympathetic nervous system, promoted the expansion of MDSCs and enhanced the immunosuppressive activities of MDSCs. Therefore, our results suggested that MDSCs may represent a novel target for pharmacologic intervention in psychological diseases. NE has been reported to have broad immunomodulatory effects. Reports shown that NE modulates cellular activity by binding to adrenergic receptors expressed on various types of cells, such as T and B cells21,22. Moreover, NE can regulate immune cell function during infection and immune challenge23. In this study, we found that NE could enhance the differentiation of human MDSCs in vitro. Additionally, NE-generated MDSCs exhibited immunosuppressive functions much stronger than control MDSCs, and this effect of NE was dependent on ROS production. We also showed that the enhanced ROS production in NE-generated MDSCs was associated with the induction of the NADPH subunit p47phox. This result was consistent with a previous report, which showed that NE increases NADPH oxidase-derived superoxide in human PBMCs24. Given that the studies have reported that MDSCs are believed to be one of the key brakes of immune system function. In our study, our data shown that NE enhanced the differentiation of human MDSCs in vitro, providing important insights into the novel roles of neurotransmitters in the regulation of myeloid cell differentiation and function. Anti-depressant therapy usually inhibits neurotransmitter release, just as NE. We hypothesized that anti-depressant drugs inhibit the release of NE, thereby reduce the expansion of MDSCs, eventually lead to the recovery of the body’s immune response. Therefore, we speculate that MDSCs may represent the potential targets and prognostic indicators of anti-depressant treatment. Of course, the next step we need to analysis is abundant clinical samples to confirm this hypothesis. In our study, we used PBMCs as our cellular model since these cells mimic the physiological hematopoietic environment in circulation; this allowed us to analyze crosstalk between neurotransmitters and immune cells25. Thus, functionally, the expansion of MDSCs induced by NE, as observed from these heterogeneous cell populations, was considered to be more representative of an intact system than analysis of individual populations of cells. Furthermore, it is important to note that these mutual effects between neurotransmitters and immune cells were observed in primary human cells and not from an immortalized cell line, providing a foundation for future patient studies to examine cellular mechanisms and potentially elucidate biomarkers for health and disease26. However, it is still unclear whether NE induces the expansion and functional activation of MDSCs in vivo; thus, additional studies are required to investigate this topic. In summary, our study showed that NE induced the expansion and activation of MDSCs, suppressing immune system function. These data provide important insights into the immunomodulatory effects of NE. Meanwhile, our findings may also raise concerns regarding the potential immunoregulatory effects of NE by facilitating the differentiation and activation of MDSCs.
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Declaration of interest The authors have no conflicts of interest to declare. This work was supported by the following grants to J.Z: National Key Basic Research Program of China (No. 2012CB524900), Guangdong Innovative Research Team Program (No. 2009010058), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (GDUPS, 2014), Program for the 12th Five-year Plan (No. 2012ZX10001003), National Natural Science Foundation of China (No. 81072397; No. 31270921), Natural Science Foundation of Guangdong (No. S2011020006072), the Fundamental Research Funds for the Central Universities, the Provincial Talents Cultivated by ‘‘Thousand-HundredTen’’ program of Guangdong Province, 111 Project (No. B12003).
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Supplementary materials are available in on-line Supplemental Figures 1 and 2
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