Increased abundance of myeloid-derived suppressor cells and Th17 cells in peripheral blood of newly-diagnosed Parkinson’s disease patients

Accepted Manuscript Title: Increased abundance of myeloid-derived suppressor cells and Th17 cells in peripheral blood of newly-diagnosed Parkinson’s disease patients Authors: Siyuan Chen, Yueqin Liu, Yuanyuan Niu, Yuhao Xu, Qianwen Zhou, Xiujian Xu, Jia Wang, Ming Yu PII: DOI: Reference:

S0304-3940(17)30275-6 http://dx.doi.org/doi:10.1016/j.neulet.2017.03.045 NSL 32733

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

4-9-2016 21-3-2017 26-3-2017

Please cite this article as: Siyuan Chen, Yueqin Liu, Yuanyuan Niu, Yuhao Xu, Qianwen Zhou, Xiujian Xu, Jia Wang, Ming Yu, Increased abundance of myeloid-derived suppressor cells and Th17 cells in peripheral blood of newly-diagnosed Parkinson’s disease patients, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.03.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Increased abundance of myeloid-derived suppressor cells and Th17 cells in peripheral blood of newly-diagnosed Parkinson's disease patients Siyuan Chena, Yueqin Liub, Yuanyuan Niua, Yuhao Xua, Qianwen Zhoua, Xiujian Xua, Jia Wangc, #, Ming Yua, # a

Department of Neurology, Affiliated Hospital of Jiangsu University, Zhenjiang,

Jiangsu, China, 212001 b

The Central Laboratory, the Fourth Affiliated Hospital of Jiangsu University,

Zhenjiang, Jiangsu, China, 212001 c

Department of Immunology, Jiangsu University. 301 Xuefu Road, Zhenjiang, Jiangsu,

China, 212013

#Corresponding

authors

Ming Yu Department of Neurology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China, 212001 Tel: 86-13861391699 E-mail: [email protected]

1

Highlights:  For the first time, increased levels of MDSCs were measured in early stage PD.  This highlights the role of Th17 cells and MDSCs in the neuroinflammatory response.  Foundation for elucidating interactions between MDSCs and Th17 cells in PD was laid.

Abstract Parkinson’s disease (PD) is a neurodegenerative disease that is often associated with corresponding neuroinflammation. In the present study, flow cytometry was used to detect T-helper 17 (Th17) cells and myeloid-derived suppressor cells (MDSCs) in 18 patients newly diagnosed with PD as well as 18 normal controls. Results showed that Th17 cells and MDSCs were significantly higher in peripheral blood of PD patients compared to controls (P < 0.001). Furthermore, there was no correlation between Th17 cells and MDSCs in peripheral blood of PD patients. Our findings suggest that Th17 cells and MDSCs may be important factors related to the occurrence and progression of PD, as well as the development of PD-related neuroinflammation. Keywords: Parkinson’s

disease;

T-helper

17

cells;

neuroinflammation

2

myeloid-derived

suppressor

cells;

1. Introduction Parkinson’s

disease

(PD)

is

a

progressive

neurodegenerative

disease

characterized by the death and degeneration of dopaminergic neurons of the substantia nigra [1]. Nearly 5 million people worldwide are afflicted with PD, and this number is likely to increase in next 20 years [2]. The onset of PD has long been associated with neuroinflammation and oxidative stress, as well as various environmental factors [3]. Recent evidence has shown that neuroinflammation plays an important role in the pathogenesis of PD. Specifically, neuroinflammation leads to excessive activation of microglia in the brain, which in turn aggravates the pathological processes of PD [4, 5]. T-helper 17 (Th17) cells are a subset of CD4+T lymphocytes that were first identified at the end of the last century [6]. Generally, Th17 cells elicit proinflammatory responses and play a major role in several inflammatory and autoimmunity diseases [7–9], with recent evidence indicating that Th17 cells are actively involved in the development of PD [6]. Myeloid-derived suppressor cells (MDSCs) are known to play crucial roles in the pathogenesis of cancers, chronic infections and inflammatory diseases. In humans, MDSCs are commonly defined as CD14-CD11b+ cells; however, these cells express the common myeloid marker CD33 but lack the expression of HLA-DR. Therefore, CD33+HLA-DR- cells have also been defined as MDSCs [10]. MDSCs have been found to inhibit inflammation by suppressing CD4+T cell activation [11]. Numerous experiments have shown that increased numbers of MDSCs were detected in myelodysplastic syndromes, tumors and inflammatory diseases [12–14]. To date, the effects of MDSCs on CD4+T cells, especially Th17 cells, remain unclear in PD [15]. As the occurrence and progression of PD are related to the development of nerve inflammation [16] and as Th17 cells 3

and MDSCs both play important roles in various diseases, such as inflammatory and autoimmune diseases [17, 18], a study was initiated to detect Th17 cells and MDSCs in peripheral blood of newly diagnosed PD patients and determine the clinical relevance between Th17 cells and MDSCs in PD. 2. Materials and methods 2.1. Patients assessment and grouping A total of 18 incipient PD patients were diagnosed from Affiliated Hospital of Jiangsu University, Zhenjiang First People’s Hospital and Zhenjiang Fourth People’s Hospital (from December 2014 to December 2015). PD severity and symptoms were evaluated using Hoehn and Yahr classification (H&Y) and the Unified Parkinson’s Disease Rating Scale (UPDRS). Inclusion criteria: Patients cooperated well in the neuropsychiatric assessment; they had good nutritional status and were without any abnormalities in blood, glucose, lipid, liver and renal functions. Exclusion criteria: Patient symptoms resulted from essential tremor, parkinsonism-plus, secondary Parkinson syndrome or hereditary Parkinson degeneration syndrome; PD patients who recently had tumors, and either immune or inflammatory diseases; patients that never received anti-PD medication. This study was approved by the Medical Ethics Committee of Affiliated Hospital of Jiangsu University, and peripheral blood samples were obtained after signing the consent form. Eighteen age- and sex-matched volunteers were enrolled as controls. 2.2. Flow cytometry analysis 2.2.1. Detection of Th17 cells in peripheral blood For detection of Th17 cells, peripheral blood was collected and stored in tubes containig heparin. Cells (2×106 /mL) were incubated in RPMI 1640 with ionomycin (500 ng/mL) and PMA (50 ng/mL; Sigma-Aldrich, Saint Louis, MO, USA). 4

GolgiPlug™ (1 µg/mL; Becton Dickinson Biosciences, San Jose, CA, USA) was added at the beginning of the culture process and cells were incubated at 37°C in 5% CO2 for 4–6 h. Stimulated cells were washed with PBS and stained with APC-CD3, FITC-CD8 and PE-IL-17A antibodies (BD PharMingen, San Diego, CA, USA), then fixed and permeabilized with Cell Fixation & Permeabilization Kit (BD PharMingen). Isotype control antibodies were used in all cases. After staining and washings, cells were resuspended and analyzed by FACSCalibur (Becton Dickinson Biosciences, Shanghai, China) equipped with FlowJo software. For gating purposes, CD4+ cells were identifed as CD3+CD8- cells and CD4+ cells expressing interleukin (IL)-17 were identified as Th17 cells. 2.2.2. Detection of MDSCs in peripheral blood For detection of MDSCs, whole blood (EDTA was used as an anticoagulant) was processed within 2 h after collection. Cells were isolated by ficoll-hypaque density gradient centrifugation and stained with PE-HLA-DR and FITC-CD33 antibodies (BD PharMingen). Isotype control antibodies were used in all cases. After staining and washings, cells were analyzed by FACSCalibur (Becton Dickinson Biosciences) equipped with FlowJo software. For gating purposes, MDSCs were identifed as CD33+HLA-DR- cells. 2.3. Statistical analyses SPSS 22.0 statistical software was used for statistical analysis and Graph Pad Prism 5 was used for the production of figures. Experimental data were represented as mean ± standard deviation. Differences between groups were determined using the Mann-Whitney U test. Correlations between two variables were quantified by determining the Spearman’s rank correlation coefficients. P < 0.05 was considered statistically significant. 5

3. Results 3.1. Patients There were no significant differences (P > 0.05) between PD patients and controls in age, gender, hemoglobin levels, white blood cell count, or in the levels of monocytes, neutrophils and lymphocytes in peripheral blood. There is no correlation between Th17 cells/MDSCs and H&Y, UPDRS or other clinical parameters (P > 0.05). Based on PD severity and symptoms (H&Y and UPDRS scores), patients were diagnosed with mild to moderate PD. Data were presented as number and mean ± standard deviation (Table. 1) 3.2. Detection of Th17 cells and MDSCs Th17 cell levels in peripheral blood of PD patients were significantly higher than those of the control group (2.34 ± 1.22 × 104 /mL, 1.17 ± 0.61% in the PD group; 0.38 ± 0.20 × 104 /mL, 0.19 ± 0.10 % in the control group; P < 0.001) (Fig. 1 and Table. 1). MDSC levels in peripheral blood of PD patients were significantly higher than those of the control group (18.72 ± 6.64 × 104/mL, 9.36 ± 3.32 % in the PD group; 3.96 ± 2.22 × 104/mL, 1.98 ± 1.11 % in the control group; P < 0.001) (Fig. 2 and Table. 1). 3.3. Correlation between Th17 cells and MDSCs Results showed there was no correlation between Th17 cells and MDSCs in peripheral blood of the PD group (r = -0.001, P > 0.05) (Fig. 3A) and the control group (r = 0.170, P > 0.05) (Fig. 3B). 4. Discussion A variety of inflammatory cytokines such as IFN-γ, IL-6 and IL-1β are released when microglia and astrocytes are excessively activated in patients with PD, leading 6

to neuroinflammation in the brain parenchyma, death of dopaminergic neurons, and a general aggravation of the pathological processes of PD [19]. Brochard reported that CD4+T lymphocytes infiltrated the substantia nigra of PD patients, suggesting that immune cells, such as T lymphocytes, were involved in the pathogenesis of PD [20]. Due to the presence of the blood brain barrier (BBB), peripheral immune cells do not enter into the brain parenchyma to mediate neuroinflammation. However, it has recently been shown that breakdown of the BBB may be a consequence of neuroinflammation [21] and furthermore, BBB breakdown has been observed in PD [22]. Interestingly, recent studies have shown that T lymphocytes may enter the central nervous system via the damaged BBB and enhance the immune response in the brain [23, 24]. For example, IL-17 and IL-22 which were released by Th17 may disrupt BBB. Furthermore, Th17 lymphocytes transmigrate across BBB, highly express granzyme B, kill neurons and promote neuroinflammatory [25]. It has been confirmed that Th17 cells play a critical role in the neuroinflammatory response in several diseases [26, 27] and in particular, these cells and the other cells such as γδT cells are known to secrete IL-17 [28, 29]. Furthermore, IL-17 is known to postivitely mediate secretion of micrgoglial-derived TNF-α and IL-1, as well as a variety of other inflammatory factors [28]. MDSCs, as a heterogeneous population, consisted of early myeloid progenitors and precursors of granulocytes, macrophages and dendritic cells [30]. CD14+HLA-DR- MDSCs and CD14+HLA-DR+ monocytes, both of which have been shown to drive the differentiation of naïve CD4+T cells [31]. For example, CD14+HLA-DR+ monocytes induce the differentiation of Th17 cells, and thus indirectly promote neuroinflammation. Furthermore, CD14+HLA-DR- MDSCs were shown to induce the amplification of regulatory T cells (Tregs) cells, a subset of CD4+T lymphocytes that mediate immune homeostasis and tolerance, which 7

negatively regulate neuroinflammation. MDSCs were shown to affect Th17 and Tregs cells in an inflammatory microenvironment, suggesting that MDSCs may regulate the balance between Th17 and Tregs cells and thus mediate the immune function in the body [32]. As the compounds used to produce PD medications may have an impact on the differentiation of Th17 cells [33], newly-diagnosed PD patients with no prior treatment were enrolled in the current study. Although correlations were made with Th17 cells and disease severity and MDSCs and disease severity, no correlations were found with disease progression, which may have been caused by insufficient numbers of patients. In the present work, we chose IL-17+CD3+CD8-cells as Th17 cells. Of course, γδT and NKT cells were also possibly included, but the frequency of γδT and NKT cells was too low in peripheral blood. Therefore, we ignore them. This study proved that Th17 cells and MDSCs in peripheral blood of PD patients were significantly higher than those in the control group, suggesting that Th17 cells and MDSCs were associated with neuroinflammation. Evidence has shown that IL-6, TGF-β, and IL-1 are significantly increased in cases of PD [34, 35] and MDSCs could produce IL-6 and TGF-β, which drives the Th17 differentiation [17, 36, 37]. In the present study, results indicated that there was no correlation between the numbers of MDSCs and Th17 cells in PD patients due to insufficient numbers of patients, which is not in agreement with previous studies examining this correlation in other diseases, including metastatic colorectal cancer [38]. In summary, Th17 cells and MDSCs participate in promoting neuroinflammatory processeses associated with PD. However, excactly how Th17 cells and MDSCs interact, and the precise role this interaction has in the progression of PD warrants 8

further investigation. Although there were increased numbers of Th17 cells and MDSCs in the peripheral blood of patients with early stage PD in this study, we could not determine whether Th17 cells or MDSCs were positively correlated with UPDRS or other clinical parameters. Furthermore, there was no direct evidence that indicated MDSCs promoted increased numbers of Th17 cells. Overall, the results suggested that MDSCs may be a critical induction factor for accumulation of Th17 cells. Taken together, the results of the current study lay the foundation for future work elucidating the precise interactions between MDSCs and Th17 cells, as well as the role this interaction has in the progression of PD. Conflicts of interest The authors declare no conflict of interest. Acknowledgments The present study was supported by Six Talent Peaks Project of Jiangsu Province (WSN038), 333 Project of Jiangsu Province (BRA2014173), Social Development Project of Zhenjiang (SH2015024, SH2015047). We thank the patients and their families for their participation in this project.

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Figure legends Fig. 1. Th17 cells in the peripheral blood from patients in the PD and control groups were detected by flow cytometry. Results showed that Th17 cells in peripheral blood of the PD group were significantly higher than those of the control group (2.34 ± 1.22 × 104 /mL, 1.17 ± 0.61% in the PD group; 0.38 ± 0.20 × 104 /mL, 0.19 ± 0.10 % in the control group, P < 0.001). LYMP: lymphocyte. The line represents the mean number.

Fig. 2. MDSCs in the peripheral blood from patients in the PD and control group were detected by flow cytometry. Results showed that MDSCs in peripheral blood of the PD group were significantly higher than those of the control group (18.72 ± 6.64 × 104/mL, 9.36 ± 3.32 % in the PD group; 3.96 ± 2.22 × 104/mL, 1.98 ± 1.11 % in the control group, P < 0.001). The line represents the mean number.

Fig. 3. There is no correlation between Th17 cells and MDSCs in peripheral blood of (A) the PD group (r = -0.001, P > 0.05) and (B) the control group (r = 0.170, P > 0.05).

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Table Table. 1 Clinical and demographic data of patients in the PD and control group. Characteristics or stages

PD group

Control group

P value

Gender male/female

9/9

9/9

1.00a

Age (range) HB (g/L)

68.28 ± 5.91 (61-79) △

126.28 ±18.89

WBC (109 /L)

5.46 ± 0.96

NEU%

59.94 ± 7.26

LYMPH%

29.42 ± 7.27

0.090b

6.16 ± 1.61

0.864b

59.93 ± 6.11

0.730b

29.59 ± 5.92

0.817b

7.52 ± 2.14

0.158b

△

△

△

7.34 ± 1.78

H&Y

1.14 ± 0.29

UPDRS-I

3.06 ± 1.11

UPDRS-II

9.39 ± 4.47

UPDRS-III

15.50 ± 6.49

UPDRS-IV

5.50 ± 2.29

0.486b

128.06 ±18.16

△

MONO (109 /L)

UPDRS-Total

64.00 ± 6.98 (51-75)

△

N/A

△

N/A

△

N/A

△

N/A

△

N/A

△

N/A

33.44 ± 13.23

Th17 cells (× 104 /mL)

2.34 ± 1.22

0.38 ± 0.20

< 0.001c

Th17%

1.17 ± 0.61

0.19 ± 0.10

< 0.001c

18.72 ± 6.64

3.96 ± 2.22

< 0.001c

9.36 ± 3.32

1.98 ± 1.11

< 0.001c

MDSCs (×104 /mL) MDSCs%

PD: Parkinson’s disease. HB: hemoglobin. WBC: white blood cell counts. NEU%: the percentage of neutrophils. LYMPH%: the percentage of lymphocytes. MONO: monocytes. H&Y: Hoehn and Yahr classification. UPDRS I, II, III, IV and Total: Unified Parkinson’s Disease Rating Scale I, II, III, IV and Total. N/A: Not applicable. Tests performed in the table included: c

Mann-Whitney U test.

△

a

chi-square,

b

Student’s t-test and

P > 0.05, there was no correlation between Th17 cells and

MDSCs in the PD group. Data were presented as number or mean ± standard deviation.

17