Expansion of polymorphonuclear myeloid-derived suppressor cells in patients with end-stage renal disease may lead to infectious complications

Myeloid-derived suppressor cells (MDSCs) are recently identified immune suppressive cells in multiple chronic inflammations. Here, we investigated MDSCs in patients with end-stage renal disease (ESRD) and their clinical significance in these patients and healthy individuals (49 each). Polymorphonuclear and mononuclear MDSCs were investigated by flow cytometry. Patients with ESRD before hemodialysis presented a significantly higher level of polymorphonuclear MDSCs. Depletion of polymorphonuclear-MDSCs resolved T cell IFN-γ responses. By co-culture, T cell proliferation and the production of IFN-γ were abrogated by the addition of polymorphonuclear MDSCs in a dose-dependent manner. Both of these effects were reversed by a reactive oxygen species inhibitor. The levels of reactive oxygen species were higher in polymorphonuclear MDSCs derived from patients with ESRD than from normal individuals. The mRNA level of NOX2, the key protein complex responsible for reactive oxygen species production, was higher in ESRD-related polymorphonuclear MDSCs. The phospho-STAT3 level, a key activator of MDSCs, was higher in ESRD-related polymorphonuclear MDSCs. Finally, the polymorphonuclear MDSC level before and after hemodialysis was positively related to infectious diseases. Patients with ESRD were dichotomized into 2 groups by the amount of polymorphonuclear MDSCs. Patients with high levels of polymorphonuclear MDSCs presented with a higher incidence of infectious events. Thus, polymorphonuclear MDSCs were elevated in ESRD patients with strong immune-suppressive capability through a phospho-STAT3/reactive oxygen species pathway. Hence, polymorphonuclear MDSCs might increase the risk of infectious complications. Myeloid-derived suppressor cells (MDSCs) are recently identified immune suppressive cells in multiple chronic inflammations. Here, we investigated MDSCs in patients with end-stage renal disease (ESRD) and their clinical significance in these patients and healthy individuals (49 each). Polymorphonuclear and mononuclear MDSCs were investigated by flow cytometry. Patients with ESRD before hemodialysis presented a significantly higher level of polymorphonuclear MDSCs. Depletion of polymorphonuclear-MDSCs resolved T cell IFN-γ responses. By co-culture, T cell proliferation and the production of IFN-γ were abrogated by the addition of polymorphonuclear MDSCs in a dose-dependent manner. Both of these effects were reversed by a reactive oxygen species inhibitor. The levels of reactive oxygen species were higher in polymorphonuclear MDSCs derived from patients with ESRD than from normal individuals. The mRNA level of NOX2, the key protein complex responsible for reactive oxygen species production, was higher in ESRD-related polymorphonuclear MDSCs. The phospho-STAT3 level, a key activator of MDSCs, was higher in ESRD-related polymorphonuclear MDSCs. Finally, the polymorphonuclear MDSC level before and after hemodialysis was positively related to infectious diseases. Patients with ESRD were dichotomized into 2 groups by the amount of polymorphonuclear MDSCs. Patients with high levels of polymorphonuclear MDSCs presented with a higher incidence of infectious events. Thus, polymorphonuclear MDSCs were elevated in ESRD patients with strong immune-suppressive capability through a phospho-STAT3/reactive oxygen species pathway. Hence, polymorphonuclear MDSCs might increase the risk of infectious complications. Infectious diseases are the major causes of death for end-stage renal disease (ESRD) patients with AIDS as 1 of the critical causes.1Taylor S.P. Taylor B.T. Health care-associated pneumonia in haemodialysis patients: clinical outcomes in patients treated with narrow versus broad spectrum antibiotic therapy.Respirology. 2013; 18: 364-368Crossref PubMed Scopus (20) Google Scholar, 2Guo H. Liu J. Collins A.J. Foley R.N. Pneumonia in incident dialysis patients–the United States Renal Data System.Nephrol Dial Transplant. 2008; 23: 680-686Crossref PubMed Scopus (65) Google Scholar Chronic kidney disease (CKD) results in accumulation of low–molecular mass metabolites, including phenylacetic acid, homocysteine, various sulfates, guanidine compounds, and others, which have inhibitory effects on immune cell activation, promote leukocyte apoptosis, and induce the oxidative burst in phagocytes.3Cohen G. Horl W.H. Immune dysfunction in uremia—an update.Toxins (Basel). 2012; 4: 962-990Crossref PubMed Scopus (112) Google Scholar, 4Kurts C. Panzer U. Anders H.J. Rees A.J. The immune system and kidney disease: basic concepts and clinical implications.Nat Rev Immunol. 2013; 13: 738-753Crossref PubMed Scopus (442) Google Scholar Recently, novel mechanisms have come to light, including elevation of Treg cells, activation of T cells, and neutrophil responsiveness to a bacterial challenge.5Hope C.M. Grace B.S. Pilkington K.R. et al.The immune phenotype may relate to cancer development in kidney transplant recipients.Kidney Int. 2014; 86: 175-183Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 6Viana J.L. Kosmadakis G.C. Watson E.L. et al.Evidence for anti-inflammatory effects of exercise in CKD.J Am Soc Nephrol. 2014; 25: 2121-2130Crossref PubMed Scopus (110) Google Scholar However, few therapies have been developed to counteract the immune deficiency of patients with ESRD, partially due to lack of practical targets. Myeloid-derived suppressor cells (MDSCs) are recently identified immune suppressive cells with the ability to suppress T cell activation and function.7Bronte V. Brandau S. Chen S.H. et al.Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.Nat Commun. 2016; 7: 12150Crossref PubMed Scopus (1566) Google Scholar, 8Nagaraj S. Schrum A.G. Cho H.I. et al.Mechanism of T cell tolerance induced by myeloid-derived suppressor cells.J Immunol. 2010; 184: 3106-3116Crossref PubMed Scopus (309) Google Scholar MDSCs were investigated initially in malignant diseases.9Talmadge J.E. Gabrilovich D.I. History of myeloid-derived suppressor cells.Nat Rev Cancer. 2013; 13: 739-752Crossref PubMed Scopus (837) Google Scholar In recent years, it has become clear that MDSCs also play an important role in the regulation of immune responses in chronic inflammations.10Parekh V.V. Wu L. Olivares-Villagomez D. et al.Activated invariant NKT cells control central nervous system autoimmunity in a mechanism that involves myeloid-derived suppressor cells.J Immunol. 2013; 190: 1948-1960Crossref PubMed Scopus (47) Google Scholar, 11Boros P. Ochando J. Zeher M. Myeloid derived suppressor cells and autoimmunity.Hum Immunol. 2016; 77: 631-636Crossref PubMed Scopus (54) Google Scholar, 12Park M.J. Lee S.H. Kim E.K. et al.Myeloid-derived suppressor cells induce the expansion of regulatory B cells and ameliorate autoimmunity in the Sanroque mouse model of systemic lupus erythematosus.Arthritis Rheumatol. 2016; 68: 2717-2727Crossref PubMed Scopus (86) Google Scholar ESRD entails chronic inflammation.3Cohen G. Horl W.H. Immune dysfunction in uremia—an update.Toxins (Basel). 2012; 4: 962-990Crossref PubMed Scopus (112) Google Scholar However, the role of MDSCs in ESRD has not been illustrated. Moreover, a series of targeted therapies have been identified to be effective in eliminating MDSCs,9Talmadge J.E. Gabrilovich D.I. History of myeloid-derived suppressor cells.Nat Rev Cancer. 2013; 13: 739-752Crossref PubMed Scopus (837) Google Scholar, 13Iclozan C. Antonia S. Chiappori A. et al.Therapeutic regulation of myeloid-derived suppressor cells and immune response to cancer vaccine in patients with extensive stage small cell lung cancer.Cancer Immunol Immunother. 2013; 62: 909-918Crossref PubMed Scopus (221) Google Scholar which made MDSC a potentially practical target for immune therapy in ESRD patients. MDSCs are now divided into 2 major populations: granulocytic or polymorphonuclear MDSC (PMN-MDSC) and mononuclear MDSC (M-MDSC).7Bronte V. Brandau S. Chen S.H. et al.Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.Nat Commun. 2016; 7: 12150Crossref PubMed Scopus (1566) Google Scholar, 9Talmadge J.E. Gabrilovich D.I. History of myeloid-derived suppressor cells.Nat Rev Cancer. 2013; 13: 739-752Crossref PubMed Scopus (837) Google Scholar, 14Condamine T. Mastio J. Gabrilovich D.I. Transcriptional regulation of myeloid-derived suppressor cells.J Leukoc Biol. 2015; 98: 913-922Crossref PubMed Scopus (197) Google Scholar In the present study, we investigated the 2 subtypes of MDSC in patients with ESRD and further illustrated their clinical significance. During the period between October 2015 and September 2016, we investigated a series of 49 patients with ERSD before initiation of dialysis in the Third Affiliated Hospital of Guangzhou Medical University and the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China (Supplementary Table S1). The ESRD was considered irreversible by 2 independent nephrologists, rather than representing acute kidney injury.6Viana J.L. Kosmadakis G.C. Watson E.L. et al.Evidence for anti-inflammatory effects of exercise in CKD.J Am Soc Nephrol. 2014; 25: 2121-2130Crossref PubMed Scopus (110) Google Scholar Age- and gender-matched healthy controls (n = 49) consisted of local volunteers. Blood samples were collected at diagnosis of ESRD and at the pre-dialysis time points under the maintenance hemodialysis phase after a median of 8-month hemodialysis (HD) vintage. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll centrifugation and analyzed within 6 hours of blood sampling. Circulating frequencies of the subsets of MDSCs were quantified with the gating strategy indicated. Both the PMN-MDSCs and M-MDSCs were CD33 positive (Figure 1a ). Patients with ESRD before dialysis presented with a significantly higher level of PMN-MDSCs, whereas the frequency of the M-MDSCs was not higher than that in healthy controls. Interestingly, after HD, the level of PMN-MDSCs decreased significantly (Figure 1b). MDSCs are characterized by their suppressive capability on T cell response because they share the same definitive markers as their normal counterparts, PMN-MDSCs and M-MDSCs.7Bronte V. Brandau S. Chen S.H. et al.Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.Nat Commun. 2016; 7: 12150Crossref PubMed Scopus (1566) Google Scholar In order to investigate whether PMN-MDSCs and M-MDSCs in patients with ESRD suppress T cell response, PMN-MDSCs and M-MDSCs were removed by flow sorting, then PBMCs and PBMCΔPMN-MDSC/M-MDSC were stimulated with anti-CD3 and anti-CD28 for 4 days. The expression of interferon-γ (IFN-γ) by CD4 and CD8 T cells was tested to evaluate T cell response.15Pallett L.J. Gill U.S. Quaglia A. et al.Metabolic regulation of hepatitis B immunopathology by myeloid-derived suppressor cells.Nat Med. 2015; 21: 591-600Crossref PubMed Scopus (185) Google Scholar As expected, depletion of PMN-MDSC and M-MDSC did not change T cell response in healthy donor–derived PBMCs (Figure 2a and b ), nor did depletion of M-MDSCs in patients with ESRD (Figure 2c). T cell IFN-γ responses were rescued by depletion of PMN-MDSCs (Figure 2d). In order to confirm the immune suppressive capacity of PMN-MDSCs in patients with ESRD, T cells and PMN-MDSCs were purified from PBMC using flow sorting. Carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled PBMC-derived CD3+ T cells were stimulated with anti-CD3 and anti-CD28, with the indicated ratio of PMN-MDSCs. CD4+ and CD8+ T cell proliferations were almost completely abrogated by the addition of ESRD-related PMN-MDSCs at a 2:1 ratio. The addition of ESRD-related PMN-MDSCs resulted in significantly reduced proliferation of both CD4+ and CD8+ T cells in a dose-dependent manner. The IFN-γ levels in the media were tested using enzyme-linked immunosorbent assay (ELISA), which determined that IFN-γ secretion decreased after administration of ESRD-related PMN-MDSCs (Figure 3). The same cells from healthy donors did not exhibit a suppressive function, indicating that PMN-MDSCs exist in patients with ESRD rather than in healthy donors. Based on the finding that PMN-MDSCs from patients with ESRD suppressed antigen-nonspecific T cell proliferation, we further explored the underlying mechanisms controlling PMN-MDSC–mediated T cell suppression. Previous reports had confirmed that production of arginase I or reactive oxygen species (ROS) were immune mediators for PMN-MDSC–mediated immune suppression.8Nagaraj S. Schrum A.G. Cho H.I. et al.Mechanism of T cell tolerance induced by myeloid-derived suppressor cells.J Immunol. 2010; 184: 3106-3116Crossref PubMed Scopus (309) Google Scholar, 14Condamine T. Mastio J. Gabrilovich D.I. Transcriptional regulation of myeloid-derived suppressor cells.J Leukoc Biol. 2015; 98: 913-922Crossref PubMed Scopus (197) Google Scholar Thus, we utilized the arginase inhibitor N(omega)-hydroxy-nor-l-arginine (nor-NOHA), L-arginine supplementation, or ROS inhibitor N-acetyl-L-cysteine (NAC) to reverse the suppressive effects of PMN-MDSCs on T cell proliferation in a co-culture system. As a result, the suppression of T cell proliferation and IFN-γ production was reversed by NAC, while nor-HOHA and L-arginine did not make a difference (Figure 4a–c ). Then, we investigated the ROS level of PMN by 2′,7′-dichlorofluorescein diacetate, which indicated that the ROS levels were higher in ESRD-related PMN-MDSCs than in healthy controls (Figure 4d). In addition, the mRNA level of the NADPH oxidase NOX2, the key protein complex responsible for ROS production in MDSCs, was higher in ESRD-related PMN-MDSCs (Figure 4e). Conversely, the expression of arginase I and the activity of arginase were similar in ESRD-related PMN-MDSCs and healthy controls (Figure 4f). According to the previous reports, p-STAT3 activation has been considered critical in MDSC activation.14Condamine T. Mastio J. Gabrilovich D.I. Transcriptional regulation of myeloid-derived suppressor cells.J Leukoc Biol. 2015; 98: 913-922Crossref PubMed Scopus (197) Google Scholar We tested the p-STAT3 level in ESRD-related PMN-MDSCs and found that it was much higher than that in healthy controls (Figure 4g). Elevated PMN-MDSCs potentially attenuated the immunity of patients with ESRD according to the results presented above. We then investigated the clinical significance of PMN-MDSCs in increasing the risk of infectious disease among patients with ESRD. First, we analyzed the association between the incidence of infectious events per month of each patient and the MDSC level and found that PMN-MDSCs before dialysis were positively associated with risk of infection episodes (Figure 5a ). Then, PMN-MDSC level at the pre-dialysis time points under the maintenance HD phase after a median of 8-month HD vintage were analyzed similarly. Interestingly, PMN-MDSCs after HD were also positively associated with risk of infection episodes (Figure 5b). Furthermore, according to the highest PMN-MDSC level (3%) in healthy controls, PMN-MDSC levels were dichotomized into 2 groups, the high and low levels. Patients with high levels of PMN-MDSCs both before and after HD presented with a critically higher incidence of infectious events (P < 0.0001) (Figure 5c). Patients with ESRD with different levels of PMN-MDSCs showed similar baseline characteristics before dialysis (Supplementary Table S1). Patients undergoing HD who had high levels of PMN-MDSCs presented with older age and higher neutrophils in differential blood leukocyte counts (Supplementary Table S2). The state of reduced renal function in patients with ESRD causes critical pathology in the immune system, including both persistent systemic inflammation and acquired immunosuppression.3Cohen G. Horl W.H. Immune dysfunction in uremia—an update.Toxins (Basel). 2012; 4: 962-990Crossref PubMed Scopus (112) Google Scholar, 4Kurts C. Panzer U. Anders H.J. Rees A.J. The immune system and kidney disease: basic concepts and clinical implications.Nat Rev Immunol. 2013; 13: 738-753Crossref PubMed Scopus (442) Google Scholar, 16Pichler R, Afkarian M, Dieter BP, Tuttle KR. Immunity and inflammation in diabetic kidney disease: translating mechanisms to biomarkers and treatment targets [e-pub ahead of print]. Am J Physiol Renal Physiol. http://dx.doi.org/10.1152/ajprenal.00314.2016.Google Scholar, 17Heine G. Sester U. Sester M. et al.A shift in the Th(1)/Th(2) ratio accompanies the clinical remission of systemic lupus erythematosus in patients with end-stage renal disease.Nephrol Dial Transplant. 2002; 17: 1790-1794Crossref PubMed Scopus (28) Google Scholar, 18Chung B.H. Kim K.W. Sun I.O. et al.Increased interleukin-17 producing effector memory T cells in the end-stage renal disease patients.Immunol Lett. 2012; 141: 181-189Crossref PubMed Scopus (41) Google Scholar The immunosuppression leads to increased infectious complications, which was the leading cause of morbidity and mortality among patients with ESRD in the current study. The loss of Igs, complement factors, zinc-binding protein, and transferrin contributed to the acquired humoral and cellular immunodeficient state that predisposed patients with nephritic syndrome to bacterial infections.3Cohen G. Horl W.H. Immune dysfunction in uremia—an update.Toxins (Basel). 2012; 4: 962-990Crossref PubMed Scopus (112) Google Scholar, 16Pichler R, Afkarian M, Dieter BP, Tuttle KR. Immunity and inflammation in diabetic kidney disease: translating mechanisms to biomarkers and treatment targets [e-pub ahead of print]. Am J Physiol Renal Physiol. http://dx.doi.org/10.1152/ajprenal.00314.2016.Google Scholar Furthermore, dysfunctional phagocytes, B cells, T cells, and macrophage defects have been described in these patients.17Heine G. Sester U. Sester M. et al.A shift in the Th(1)/Th(2) ratio accompanies the clinical remission of systemic lupus erythematosus in patients with end-stage renal disease.Nephrol Dial Transplant. 2002; 17: 1790-1794Crossref PubMed Scopus (28) Google Scholar, 18Chung B.H. Kim K.W. Sun I.O. et al.Increased interleukin-17 producing effector memory T cells in the end-stage renal disease patients.Immunol Lett. 2012; 141: 181-189Crossref PubMed Scopus (41) Google Scholar, 19Snanoudj R. Claas F.H. Heidt S. et al.Restricted specificity of peripheral alloreactive memory B cells in HLA-sensitized patients awaiting a kidney transplant.Kidney Int. 2015; 87: 1230-1240Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar However, the roles of multiple immune cells were far from being identified in the immune dysfunction of patients with ESRD. In the present study, we have identified a novel immune deficiency–inducing cell group, PMN-MDSC, in patients with ESRD and indicated their contribution to infectious complications. In the present study, an elevated PMN-MDSC level was found in patients with ESRD. MDSCs, first investigated in cancer,9Talmadge J.E. Gabrilovich D.I. History of myeloid-derived suppressor cells.Nat Rev Cancer. 2013; 13: 739-752Crossref PubMed Scopus (837) Google Scholar have been found to be elevated in some chronic inflammation conditions, such as chronic hepatitis and HIV infection,15Pallett L.J. Gill U.S. Quaglia A. et al.Metabolic regulation of hepatitis B immunopathology by myeloid-derived suppressor cells.Nat Med. 2015; 21: 591-600Crossref PubMed Scopus (185) Google Scholar, 20Qin A. Cai W. Pan T. et al.Expansion of monocytic myeloid-derived suppressor cells dampens T cell function in HIV-1-seropositive individuals.J Virol. 2013; 87: 1477-1490Crossref PubMed Scopus (128) Google Scholar, 21Tumino N. Turchi F. Meschi S. et al.In HIV-positive patients, myeloid-derived suppressor cells induce T-cell anergy by suppressing CD3zeta expression through ELF-1 inhibition.AIDS. 2015; 29: 2397-2407Crossref PubMed Scopus (38) Google Scholar as well as in physiological conditions, including pregnancy and infancy.22Kostlin N. Kugel H. Spring B. et al.Granulocytic myeloid derived suppressor cells expand in human pregnancy and modulate T-cell responses.Eur J Immunol. 2014; 44: 2582-2591Crossref PubMed Scopus (112) Google Scholar, 23Gervassi A. Lejarcegui N. Dross S. et al.Myeloid derived suppressor cells are present at high frequency in neonates and suppress in vitro T cell responses.PLoS One. 2014; 9: e107816Crossref PubMed Scopus (69) Google Scholar A series of proinflammatory cytokines have been reported to induce MDSCs critically, such as IL-6, TNF-α, IL-13, and IFN-γ, which have also been found to be elevated in patients with ESRD.9Talmadge J.E. Gabrilovich D.I. History of myeloid-derived suppressor cells.Nat Rev Cancer. 2013; 13: 739-752Crossref PubMed Scopus (837) Google Scholar, 14Condamine T. Mastio J. Gabrilovich D.I. Transcriptional regulation of myeloid-derived suppressor cells.J Leukoc Biol. 2015; 98: 913-922Crossref PubMed Scopus (197) Google Scholar, 17Heine G. Sester U. Sester M. et al.A shift in the Th(1)/Th(2) ratio accompanies the clinical remission of systemic lupus erythematosus in patients with end-stage renal disease.Nephrol Dial Transplant. 2002; 17: 1790-1794Crossref PubMed Scopus (28) Google Scholar Thus, the existence of PMN-MDSCs in patients with ESRD might balance out to the proinflammatory status of patients with ESRD. None of the patients with ESRD received erythropoietin before dialysis, which eliminated its influence on PMN-MDSC level. Interestingly, PMN-MDSC level decreased significantly after a period of HD, indicating that PMN-MDSC was induced by an effect of azotemia. It is well known that PMN-MDSCs exert their suppressive capability through ROS or arginase.8Nagaraj S. Schrum A.G. Cho H.I. et al.Mechanism of T cell tolerance induced by myeloid-derived suppressor cells.J Immunol. 2010; 184: 3106-3116Crossref PubMed Scopus (309) Google Scholar, 9Talmadge J.E. Gabrilovich D.I. History of myeloid-derived suppressor cells.Nat Rev Cancer. 2013; 13: 739-752Crossref PubMed Scopus (837) Google Scholar, 11Boros P. Ochando J. Zeher M. Myeloid derived suppressor cells and autoimmunity.Hum Immunol. 2016; 77: 631-636Crossref PubMed Scopus (54) Google Scholar In the present study, multiple evidence indicates that ERSD-related PMN-MDSC suppresses T cells through ROS. Meanwhile, protein oxidation was 1 of the features of ESRD status3Cohen G. Horl W.H. Immune dysfunction in uremia—an update.Toxins (Basel). 2012; 4: 962-990Crossref PubMed Scopus (112) Google Scholar, 4Kurts C. Panzer U. Anders H.J. Rees A.J. The immune system and kidney disease: basic concepts and clinical implications.Nat Rev Immunol. 2013; 13: 738-753Crossref PubMed Scopus (442) Google Scholar that attenuated the activity of enzymes, cytokines, and antibodies, contributing to both general inflammation and immune dysfunction in patients with ESRD. Thus, elevated PMN-MDSCs inhibited the immune system as a critical source of ROS in patients with ESRD. Furthermore, p-STAT3 activation is reported to be a critical pathway for MDSC.14Condamine T. Mastio J. Gabrilovich D.I. Transcriptional regulation of myeloid-derived suppressor cells.J Leukoc Biol. 2015; 98: 913-922Crossref PubMed Scopus (197) Google Scholar In the present study, we found that p-STAT3 was activated in ESRD-related PMN-MDSC, which indicated that the p-STAT3/ROS pathway was the mechanism involved. ESRD-related PMN-MDSCs are associated with infectious complications. The present study indicates that ESRD patients with a higher level of PMN-MDSCs present with an increased risk of infectious complications, parallel with the immune suppressive function of PMN-MDSCs. This finding was reinforced by re-analysis of PMN-MDSCs during maintenance HD. Patients with higher PMN-MDSC levels before and after HD presented with a higher risk of infectious events. Notably, MDSCs have been reported to play a role in osteoclasia in the context of rheumatoid arthritis,11Boros P. Ochando J. Zeher M. Myeloid derived suppressor cells and autoimmunity.Hum Immunol. 2016; 77: 631-636Crossref PubMed Scopus (54) Google Scholar, 24Zhang H. Huang Y. Wang S. et al.Myeloid-derived suppressor cells contribute to bone erosion in collagen-induced arthritis by differentiating to osteoclasts.J Autoimmun. 2015; 65: 82-89Crossref PubMed Scopus (48) Google Scholar indicating that PMN-MDSCs might be involved in renal osteodystrophy. Thus, MDSC might be a promising target for immunotherapy in patients with ESRD, and a series of clinically available agents targeting MDSC,9Talmadge J.E. Gabrilovich D.I. History of myeloid-derived suppressor cells.Nat Rev Cancer. 2013; 13: 739-752Crossref PubMed Scopus (837) Google Scholar such as all-trans-retinoic acid and cyclooxygenase 2 (COX2) inhibitors, might benefit patients with ESRD. However, none of the patients received peritoneal dialysis or erythropoietin, and 7 of 49 patients in our study rejected dialysis due to personal reasons including financial issues (4 patients), fear of dialysis (1 patient), and rejection of lifestyle changes (2 patients). These weaknesses in patient management might increase the severity of ESRD among the patients and upregulate the PMN-MDSC levels. Patients under better management might present with decreased PMN-MDSC levels and less risk for infectious events. In addition, PMN-MDSC percentage decreased and M-MDSC percentage increased after HD, which might be caused by membrane exposure and should be investigated in future studies. In summary, the present study found that PMN-MDSCs were elevated in patients with ESRD and exhibited a strong immune-suppressive capability through the p-STAT3/ROS pathway. PMN-MDSCs might increase the risk of infectious complications and offer a promising target for immune therapy. All patients and healthy controls were screened for serum HIV antibody, hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) antibody, hepatitis D virus (HDV) antigen, and HDV antibody. Patients and healthy controls who tested positive for HIV, had chronic hepatitis virus infection or other acute infections (e.g., pneumonia or urinary tract infection), were pregnant, received systematic corticosteroids or immnunosuppressive agents, or exhibited a fever were excluded from this study. The study was approved by the Clinical Ethics Review Board of the Third Affiliated Hospital of Guangzhou Medical University. A written informed consent was obtained from all patients at the time of admission. The following anti-human antibodies were purchased from eBioscience (San Diego, CA): CD11b-fluorescein isothiocyanate (FITC), HLA-DR–APC, CD14-PE-Cy7, CD15-eFluor450, CD33-PE, and their corresponding isotype controls. pY705-Stat3-AlexaFluor 488 was purchased from BD Biosciences (San Jose, CA). The cell phenotypes were analyzed by flow cytometry on a FACSAria II flow cytometer (BD Biosciences), and data were analyzed with the FlowJo V10.0.7 (FlowJo, Ashland, OR). For the flow cytometric sorting, a BD FACSAria cell sorter (BD Biosciences) was used. The strategy for MDSC sorting was CD14–CD11b+CD15+HLA-DR–/low for PMN-MDSCs and CD11b+CD14+HLA-DR–/lowCD15– for M-MDSCs from live PBMCs.7Bronte V. Brandau S. Chen S.H. et al.Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.Nat Commun. 2016; 7: 12150Crossref PubMed Scopus (1566) Google Scholar Depletion of MDSCs was performed by harvesting the remaining PBMCs after MDSC sorting. The experiments were performed in a biosafety laboratory. T cell proliferation was determined by CFSE (5,6-carboxyfluoresceindiacetate, succinimidylester) dilution. Purified T cells were labeled with CFSE (3 μM; Invitrogen, Carlsbad, CA), stimulated with 0.5 μg/ml 3-hour pre-coated anti-CD3 and 0.5 μg/ml anti-CD28 (eBioscience), and cultured alone or co-cultured with autologous MDSCs at the indicated ratios for 3 days. The cells were then stained for surface marker expression with CD4-PE or CD8-PE-Cy5 antibodies, and T cell proliferation was analyzed on a flow cytometer. All cultures were carried out in the presence of 20 IU/ml recombinant human IL-2 (PeproTech, Rocky Hill, NJ) in RPMI 1640 (Life Technologies, Carlsbad, CA) for 3 to 4 days at 37°C. Specific responses were detected by intracellular cytokine staining after restimulation on day 4 in the presence of 1 μg/ml brefeldinA (BFA; Sigma-Aldrich, St. Louis, MO). Where indicated, 1 mM L-arginine, 0.5 mM nor-NOHA (Cayman Chemicals, Ann Arbor, MI), an arginase I–specific inhibitor, or 1 mM N-acetylcysteine (Sigma-Aldrich), an ROS inhibitor, was added to the culture on day 0. ROS measurements were evaluated using the 2′,7′-dichlorofluorescein diacetate stain. Briefly, PBMCs were incubated with 2.5 μM 2′,7′-dichlorofluorescein diacetate at 37°C for 30 minutes (min). Cells were then washed and resuspended in phosphate-buffered saline, and analyzed using flow cytometer with excitation and emission wavelengths of 490 and 520 nm, respectively. IFN-γ quantification in culture supernatants was determined using ELISA per manufacturer’s instructions (R&D Systems, Minneapolis, MN). The activity of arginase was measured in cell lysates. Briefly, cells were lysed with 0.1% Triton X-100 for 30 min, then 25 mM Tris-HCl and 10 mM MnCl2 were added. The enzyme was activated by heating at 56°C for 10 min. Arginine hydrolysis was performed by incubating the lysate with 0.5 M L-arginine for 120 min at 37°C. After the addition of α-isonitrosopropiophenone (dissolved in 100% ethanol), the urea concentration was measured at 540 nm, followed by heating at 95°C for 30 min. RNA was extracted with a Multisource Total RNA Miniprep Kit (Axygen, Union City, CA), and quantitative reverse transcription polymerase chain reaction was performed utilizing commercially available primers (Supplementary Table S3) and SYBR Premix Ex Taq II (Code, DRR081; Takara Biotechnology Co., Dalian, China). Fluorescence for each cycle was quantitatively analyzed using the ABI Prism 7000 sequence detection system (Life Technologies). The results were reported as relative expression, normalized with GAPDH housekeeping gene as an endogenous control and expressed in arbitrary units. Forty-two patients were treated with HD, and the remaining 7 patients rejected HD and died of renal failure–related complications. The baseline characteristics of patients with or without HD were similar (Supplementary Table S4). The catheter-related infection, sepsis, and major acute infectious events in the respiratory system, urinary tract, and digestive tract of each patient were noted during follow-up prospectively. Clinical and immunological parameters, including urea reduction ratio, Kt/V, ages, gender, and erythropoietin usage, were compared by nonparametric Mann-Whitney U tests or chi-square test. For in vitro experiments, statistical analyses were conducted using paired t-tests. Correlations between different parameters were analyzed using a Spearman rank test and linear regression. Statistical tests were performed using GraphPad Prism version 5.0a and SPSS Statistics 20.0. P values ≤ 0.05 were considered significant. All the authors declared no competing interests. This study was supported by National Natural Science Foundation of China (31600710), Natural Science Foundation of Guangdong (No. 2014A030313146 and 2016A030313302), Innovative Training Program of Sun Yat-sen University (201601073), and Laboratory Open Fund of Sun Yat-sen University (20160117). The authors thank Shang-Xin Liu and Wen-Hui Jiang for their contribution in sample transportation. Download .docx (.01 MB) Help with docx files Table S1Comparing characteristics of end-stage renal disease patients with low and high levels of polymorphonuclear myeloid-derived suppressor cells. Download .docx (.02 MB) Help with docx files Table S2Comparing characteristics of hemodialysis patients with low and high levels of polymorphonuclear myeloid-derived suppressor cells. Download .docx (.01 MB) Help with docx files Table S3Primer sequence used. Download .docx (.02 MB) Help with docx files Table S4Comparing characteristics of end-stage renal disease patients with and without hemodialysis.