Expansion and activation of monocytic-myeloid-derived suppressor cell via STAT3/arginase-I signaling in patients with ankylosing spondylitis
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Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease. The dysregulated immune system plays an important role in the pathogenesis of AS. Myeloid-derived suppressor cells (MDSCs) play a key immunoregulatory role in autoimmune arthritis. The aim of this study was to clarify the underlying immunoregulatory mechanism of MDSCs in patients with AS.
Flow cytometry was used to analyze the phenotype of MDSCs among peripheral blood mononuclear cells (PBMCs) from 46 patients with AS and 46 healthy control subjects. The correlation between MDSC frequency and the disease index of patients with AS was evaluated. A T cell proliferation experiment was used to evaluate the immunosuppressive function of MDSCs.
Polymorphonuclear (PMN) and monocytic (M)-MDSCs were significantly elevated in the PBMCs of patients with AS, when compared with levels in healthy controls. Additionally, M-MDSC levels correlated positively with the clinical index of AS, including the Bath ankylosing spondylitis disease activity index (BASDAI) score, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels. M-MDSCs derived from patients with AS suppressed T cell responses, and this effect was dependent on the induction of arginase-I. Furthermore, AS-derived M-MDSCs showed high levels of phosphorylated STAT3. Stattic, a STAT3-specific inhibitor, and STAT3-targeted siRNA abrogated the immunosuppressive function of M-MDSCs. Inhibition of STAT3 signaling also resulted in decreased arginase-I activity.
STAT3/arginase-I signaling plays an important role in both the expansion and activation of M-MDSCs in patients with AS. This information may be beneficial in developing novel therapeutic strategies for preventing AS.
KeywordsAnkylosing spondylitis Myeloid-derived suppressor cells STAT3/arginase-I signaling T cell suppression
Bath Ankylosing Spondylitis Disease Activity Index
Carboxyfluorescein succinimidyl ester
Enzyme-linked immunosorbent assay
Erythrocyte sedimentation rate
Immature myeloid cells
Monocytic myeloid-derived suppressor cells
Peripheral blood mononuclear cells
Polymorphonuclear myeloid-derived suppressor cells
Reactive oxygen species
Signal transducer and activator of transcription 3
Regulatory T cells
Expansion of M-MDSCs subset in PBMCs derived patients of AS.
STAT3/arginase-I pathway mediated the expansion of M-MDSC.
Ankylosing spondylitis (AS) is a chronic inflammatory disease that affects the axial skeleton, causing characteristic inflammatory back pain . The prevalence of different types of spondyloarthritis is 0.5–1.9%, and interaction between a strong genetic component, mainly by specific HLA-B27 subtypes, and bacteria seems to be crucial for the development of the disease . Although there have been significant findings in understanding the pathogenesis of AS, the exact mechanisms have not yet been identified [3, 4]. Clinical therapy and diagnosis are mainly dependent on the radiographic progression of AS . Therefore, understanding the molecular progression of AS would facilitate early diagnosis and treatment during pathogenesis. Immunohistological studies on sacroiliac joint biopsies have shown immune cell infiltrates, including T cells and macrophages, suggesting that both innate and adaptive immune responses could play a role in AS pathogenesis . Further studies determined that gut immunity, T-lymphocyte activation, and peptide processing before HLA class I presentation are involved in the pathogenesis of AS . Studies showing increased frequencies of interleukin (IL)-17-positive CD4+ T cells in peripheral blood mononuclear cells (PBMCs) obtained from patients with AS support the fact that T helper (Th)17 cells are involved in the pathogenesis of inflammatory arthritis [8, 9]. Moreover, imbalances in the T lymphocyte subset ratios, Th1/Th2 and Th17/regulatory T (Treg), were demonstrated in patients with AS . These studies collectively indicate that AS progression may be associated with the degree of immune abnormality.
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that consists of myeloid progenitor cells and immature myeloid cells (iMCs). In healthy individuals, iMCs generated in the bone marrow quickly differentiate into mature granulocytes, macrophages, or dendritic cells . In pathological conditions, such as in cancer and some autoimmune diseases, a partial block in the differentiation of iMCs into mature myeloid cells results in the expansion of the MDSC population . MDSCs constitute a unique component of the immune system that regulates immune responses in healthy individuals and in the context of various diseases [13, 14]. MDSCs are classified into two major subsets based on their phenotypic and morphological features: polymorphonuclear (PMN)-MDSCs and monocytic (M)-MDSCs . In mice, MDSCs are defined as cells expressing both Gr-1 and CD11b, and are further classified into two subpopulations based on Ly6G and Ly6C: PMN-MDSC (CD11b+ Ly6G+ Ly6Clo) and M-MDSC (CD11b+ Ly6G− Ly6Chi) . In human PBMCs, the equivalent subsets to PMN-MDSCs and M-MDSCs are defined as HLA-DRlow/− CD11b+CD33+CD14−CD15+ and HLA-DRlow/− CD11b+CD33+CD14+CD15−, respectively . MDSCs are characterized by an immunosuppressive phenotype. L-arginine metabolism plays a central role in the immunosuppressive activity of MDSCs. L-arginine can be metabolized by inducible nitric oxide synthase (iNOS or Nos2), generating citrulline and nitric oxide (NO), or can be converted into urea and L-ornithine by arginase . MDSCs expressing arginase-I (ARG1) reduce the availability of L-arginine, which can result in the loss of CD3ζ expression and impaired T cell function .
Several recent reports show that MDSCs play crucial roles in the regulation of autoimmune diseases. The MDSC population showed significant expansion in arthritic mice and in patients with rheumatoid arthritis (RA) and produced high levels of inflammatory cytokines . In addition, MDSCs from collagen-induced arthritis (CIA) model mice and patients with RA promoted the polarization of Th17 cells, displaying T cell suppressive ability . Furthermore, the transfer of these CIA mouse-derived MDSCs facilitated disease progression in CIA model mice . These studies collectively show the potential association of MDSCs with autoimmune arthritis disease as well as the therapeutic value of MDSCs. However, the association between MDSCs and AS has not been examined.
In this study, we report that MDSCs showed expansion in patients with AS compared with healthy controls, and the level of M-MDSCs significantly correlated with the AS disease activity index. AS-derived M-MDSCs displayed a T cell suppressive function, which was mediated through the production of arginase-I and activation of STAT3 (signal transducer and activator of transcription 3) signaling. Our study provides novel insights into a valuable role of M-MDSCs in promoting AS pathogenesis and suggests that M-MDSCs represent a potential immune therapeutic target in AS treatment.
This research was approved by the ethics review board of Guangdong Second Provincial General Hospital. Written, informed consent was provided by each participant and/or their legal guardian.
Characteristics of the patients with ankylosing spondylitis
Number of samples (n)
32.3 ± 1.2
23.6 ± 9.8
Gender, male/female (n)
Duration of disease (years)
5.6 ± 3.6
2.76 ± 1.07
9.2 ± 3.5
24.1 ± 12.4
2.5 ± 1.2
28 ± 17.1
HLA-B27, positive member
Reagents and antibodies
RPMI 1640, DMEM, Lipofectamine 2000, FBS, β-ME, penicillin, 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), and 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) were obtained from Invitrogen (Grand Island, NY, USA). NW-hydroxy-nor-arginine (NOHA) and L-NG-monomethyl-arginine (L-NMMA) were obtained from Cayman Chemical (Ann Arbor, MI), N-acetylcysteine (NAC) and dimethyl sulfoxide were purchased from Sigma-Aldrich (Merck, Germany). The following anti-human Abs was purchased from Thermo Fisher Scientific (Waltham, MA, USA): CD11b-FITC, CD33-PE, CD14-PE-Cy7, CD15-eFluor450, HLA-DR-PE-Cy5, CD4-PE, CD8-PE-Cy5, CD3-PE-Cy7, and their corresponding isotope controls.
PBMCs isolation and flow cytometric analysis
PBMC were isolated from whole blood by Ficoll centrifugation and analyzed immediately. The cell phenotype was analyzed by flow cytometer (BD LSR fortessa; BD Biosciences, San Jose, CA, USA), and the data were analyzed with the FlowJo 10.0 software package (TreeStar Inc., Ashland, OR, USA). Data were acquired as the fraction of labeled cells within a live-cell gate set for 50,000 events. A FACS Aria III (BD Biosciences) was used for flow cytometric sorting. The strategy for MDSCs sorting was to gate HLA-DR-/lowCD11b+CD33int/high cells from PBMC. In some experiment, MDSCs were sorted from PBMC; then, the remaining PBMC were used for the T cell proliferation assay.
T cell proliferation assay
T cell proliferation was evaluated by CFSE dilution. Purified T cells were labeled with CFSE (3 μM; Invitrogen), stimulated with antiCD3/CD28 antibodies (5 μg/ml, Thermo Fisher Scientific), and cultured alone or co-cultured with autologous PMN-MDSCs or M-MDSCs at the indicated ratios. 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. T cells without stimulation were used as the negative control.
Arginase enzymatic activity assay
Arginase-I activity was measured in PMN-MDSC lysates, as previously described  with slight modifications. Briefly, cells were lysed with 0.1% Triton X-100 for 30 min, followed by the addition of 25 mM Tris-HCl and10 mM MnCl2. The enzyme was activated by incubation for 10 min at 56 °C. Arginine hydrolysis was performed by incubating the lysate with 0.5 M l-arginine at 37 °C for 2 h. The urea concentration was measured at 540 nm after the addition of alpha-isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 95 °C for 30 min.
Reactive oxygen species (ROS) production
Cells (5 × 105) were incubated at 37 °C in the presence of 1 μM CMH2DCFDA (Thermo Fisher Scientific) for 30 min and were then labeled with fluorescence-conjugated antibodies (Abs) against CD33 and CD11b. The ROS content in PMN-MDSCs was analyzed by flow cytometry.
Enzyme-linked immunosorbent assay (ELISA)
The production of interferon (IFN)-γ in culture supernatants was determined by ELISA, following the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).
PMN-MDSCs isolated by flow cytometric sorting were cultured in Transwell inserts (0.4 mm pore size; EMD Millipore, Billerica, MA, USA), and fresh autologous T cells (1 × 106 cells/ml) were cultured in 96-well plates.
Intracellular staining of phosphorylated STAT-3 (pSTAT-3, phospho Tyr705) was performed following the manufacturer’s protocol (Cell Signaling Technology, Beverly, MA, USA). Cells (5 × 105) were fixed with formaldehyde to stabilize the cell membrane, and permeabilized using BD Fix&Perm Solution (BD Biosciences, Franklin Lakes, NJ, USA). After washing, cells were then stained with Alexa Fluor 488-conjugated pSTAT-3 and analyzed by flow cytometry.
Stattic, a STAT3-specific small molecule inhibitor (Calbiochem, MilliporeSigma, Burlington, MA, USA) and short interfering (si) RNA targeting STAT3 (siSTAT3 ID: 116558, Thermo Fisher Scientific) were used to inhibit STAT3 signaling. Stattic was diluted to 1% in dimethyl sulfoxide (DMSO). PMN-MDSCs were treated with 10 μM Stattic at 37 °C for 24 h. Scrambled and STAT3-targeted siRNAs were transduced into PMN-MDSCs cells using lentiviral vectors .
Generation of retrovirus
Two hunderd ninety three T cells were transfected with a mixture of DNA containing 2.5 μg VSVG, 2.5 μg Δ8.2, and scrambled or STAT3-targeted siRNA vectors using Lipofectamine 2000 according to the manufacturer’s instructions. Media containing scrambled or STAT3 siRNA retroviruses were collected 72 h after transfection and filtered through a 0.45 μm pore-size filter.
All data are presented as the mean ± SEM. Clinical and immunological parameters were compared by non-parametric Mann-Whitney U tests. For in vitro experiments, statistical analyses were performed using unpaired or paired t tests. Correlations between different parameters were analyzed using a Spearman rank test. Statistical tests were performed using GraphPad Prism version 5.0a (GraphPad Software, San Diego, CA, USA) and SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA). P values of 0.05 or 0.01 were considered significant.
Increased frequency of MDSCs in peripheral blood of patients with AS
Elevated M-MDSCs correlate with disease index in patients with AS
M-MDSCs derived from patients with AS suppress T cell responses
AS-derived M-MDSCs suppress T cell responses in an arginase-I-dependent manner
Inhibition of pSTAT3/arginase-I signaling abrogates the suppressive activity of AS-derived M-MDSCs
Although MDSCs have been intensively investigated in autoimmune arthritis, recent studies have shown an emerging role for MDSCs in the pathogenesis of RA . However, the mechanisms for the aberrant expansion of MDSCs in AS as well as their immunological and clinical significance remain unclear. Delineating these important issues will advance our understanding of the relationship between MDSCs and AS, which will benefit the development of immunotherapies to treat human AS diseases.
Here, we report a significant elevation of both subsets of M-MDSCs and PMN-MDSCs in the peripheral blood of patients with AS. Consistent with the CIA mice model and patients with RA, MDSCs significantly expanded in arthritic mice and patients with RA, suggesting that MDSCs play a key role in autoimmune arthritis. However, the mechanism underlying the MDSC expansion in autoimmune arthritis has not yet been determined. In CIA mice, inflammatory cytokines promote myelopoiesis by stimulating the production of myeloid precursors in the bone marrow. Increased levels of TNF-α and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) may promote MDSC accumulation in the PBMC and spleen. In addition, interleukin 6 (IL-6) and transforming growth factor-beta 1 (TGFβ1) genes most likely cause the expansion of MDSCs in inflammatory or cancerous conditions . Similarly, MDSCs from arthritic mice also express higher levels of inflammatory cytokines (e.g. TNF-α, IL-1β) than those from control mice, supporting an inflammatory activation in these cells. These studies suggest that in inflamed tissues, pro-inflammatory cytokines could promote MDSC aggregation, and in turn, MDSCs can releases more pro-inflammatory factors to aggravate inflammatory responses. Moreover, MDSCs from CIA mice and patients with RA promoted the polarization of Th17 cells in vitro . Interestingly, Th17 cells are also involved in the pathogenesis of AS . In our study, we also found that there is a positive correlation between the percentages of M-MDSCs and IL-17 levels in patients with AS. In addition to IL-17, circulating M-MDSCs are elevated significantly in patients with AS with positive correlations to elevated disease activity index, including BASDAI, ESR, and CRP. These data suggest that MDSCs play crucial roles in the regulation of AS.
MDSC-mediated suppression of T cell responses could be beneficial in pathological conditions characterized by the unopposed activation of the immune system such as autoimmune diseases . However, the therapeutic potential of MDSCs in autoimmune arthritis is contradictory. One study showed that the transfer of MDSCs derived from CIA mice and patients with RA facilitated disease progression . In contrast, another study found that the adoptive transfer of CIA-derived MDSCs could reduce the severity of CIA, and the number of Th17 cells also decreased . These inconsistent outcomes may arise from the heterogeneity of MDSCs, factors within the autoimmune inflammatory environment, and different states of the disease. Further investigations are required to study the therapeutic effect of MDSCs in an AS model through AS-derived-M-MDSC transfer, which will be beneficial in understanding the pathological mechanism of AS and provide key insights for developing MDSC-based therapies to treat AS.
MDSCs are primarily defined by their suppressive function , however, the functions of MDSCs in patients with AS remain unclear. The T cell suppressive effect of MDSCs in malignant tumors is due to M-MDSCs rather than PMN-MDSCs . However, PMN-MDSCs have been reported to show suppressive functions in autoimmune disease models . It is possible that the phenotypes of MDSCs responsible for suppressing T cell functions differ between tumors and autoimmune diseases. In our study, we found that the T cell suppressive effect of MDSCs in patients with AS is due to M-MDSCs rather than PMN-MDSCs. We examined regulatory factors in M-MDSCs that could control its suppressive function. A critical pathway in tumors and periphery is mediated by STAT3 signaling in the M-MDSC population . In murine models, pSTAT3 regulates the expansion of MDSCs; however, STAT3 has not been reported to directly regulate the T cell suppressive function of MDSCs . In contrast, the immunosuppressive activity of human MDSCs derived from patients with cancer was found to be STAT3-dependent . In this study, we demonstrated that STAT3 signaling plays a functional role in M-MDSCs derived from patients with AS by mediating their ability to suppress autologous T cell proliferation. STAT3 inhibition decreased the level of arginase-I and the T cell suppressive activity in AS-derived M-MDSCs. We also demonstrated that both siRNA suppression and pSTAT3 inhibition using a STAT3-specific inhibitor could abrogate the T cell suppressive function of AS-derived M-MDSCs as well as decrease the level and activity of arginase-I. These results demonstrate that STAT3 signaling is upstream of the arginase-I activity that mediates the suppression of T cell proliferation in patients with AS.
Several STAT3-dependent genes have been reported to play critical roles in M-MDSC function, indicating that there may be multiple pathways of STAT3-dependent immunosuppression. For instance, STAT3-dependent C/EBPβ transcription factor is critical in regulating immunosuppression . HIF1α, another STAT3-dependent gene, mediates the differentiation into tumor-infiltrating macrophages . Further investigations will be beneficial in testing some of these STAT3-dependent pathways in M-MDSCs.
We report that M-MDSCs were significantly elevated in patients with AS. M-MDSC numbers positively correlated with the AS disease index. The STAT3/arginase-I signaling pathway drove the expansion of M-MDSCs, and mediated the activation of the T cell suppressive function in AS-derived M-MDSCs. Our results collectively suggest that M-MDSCs play an important immunoregulatory role in patients with AS, and therapeutic approaches directed against M-MDSCs may lead to the alleviation of this disease.
We thank Shao-hua Song from the Department of Laboratory Medicine, Guangdong Second Provincial General Hospital, for kindly providing assistance in the clinical sample collection.
This work was supported by the National Natural Science Foundation of China (81700512) to YL; Natural Science Foundation of Guangdong Province (2016A030310252) to YL; Guandong Second Provincial General Hospital Youth Fund (YQ2016-007) to ZL.
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. FL and LZ had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. YL, LZ, and FL contributed to study conception and design. YL, KZ, HJ, XZ, BC, and PL contributed to acquisition of data. YL, LZ, and FL contributed to analysis and interpretation of data:.
Ethics approval and consent to participate
This research was approved by the ethics review board of Guangdong Second Provincial General Hospital. Written, informed consent was provided by each participant and/or their legal guardian.
Consent for publication
The authors declare that they have no competing interests.
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