Background
Systemic lupus erythematosus (SLE) is a systemic autoimmune syndrome with unclear etiology that affects multiple organs and afflicts mostly women of childbearing age. The skin, blood vessels, kidneys, central nervous system, and joints are common targets of inflammation at onset or during the course of the disease. The development of SLE is attributed to disruptions in adaptive immunity, triggered by genetic predisposing factors and environmental triggers, which lead to the loss of tolerance to self-antigens. Indeed, B and T lymphocytes play prominent pathogenic roles in the development and progression of SLE [
1,
2].
However, over the last few decades, evidence has clearly indicated the disorder of innate immune responses in the development of SLE [
3‐
6]. Neutrophils, the most abundant white blood cells in humans, play crucial roles as sentinels and first-line of defense against pathogens in the innate immune system. Recent evidence indicates that neutrophils may play an important role in the induction of autoimmune responses and organ damage in SLE [
7,
8]. Lupus neutrophils have been reported to have altered functional properties, including diminished phagocytic capabilities, aberrant clearance of apoptotic material, decreased responsiveness to cytokines, increased aggregation, and intravascular activation [
9]. Nevertheless, the role of lupus neutrophils in SLE pathogenesis has not been well-elucidated.
Programmed death ligand 1 (PD-L1) is one of the costimulatory molecules in the B7 family, which functions as an immunomodulatory molecule. The engagement of PD-L1 with its receptor, programmed death 1 (PD-1), delivers inhibitory signals to target cells such as activated T cells and B cells, and thus helps to maintain the balance between effective immunity, tolerance and immunopathology [
10]. PD-L1 is broadly expressed on a variety of immune cells, including T cells, B cells, dendritic cells, and monocytes. Recent evidence indicates that PD-L1 is also expressed on neutrophils and is associated with the development of numerous diseases, including human immunodeficiency virus [
11], sepsis [
12], Burkholderia pseudomallei-infected disease [
13], and tuberculosis [
14]. However, the frequency and roles of PD-L1-expressing neutrophils in SLE has not been established. In the present study, we determined the frequency of PD-L1-expressing neutrophils in patients with SLE and tested the hypothesis that their frequency correlates with the activity and severity of SLE.
Methods
Subjects
Patients (n = 77) who patients fulfilled the revised American College of Rheumatology criteria for SLE [
15] were enrolled from the First Affiliated Hospital of Nanchang University from June 2014 to July 2015. Disease activity was assessed by the SLE disease activity index (SLEDAI) [
16]. In addition, this study included 43 healthy controls without inflammatory or autoimmune diseases, who were unrelated to the patients. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University and was carried out in compliance with the Helsinki Declaration. Informed consent was obtained from all the participants before they entered the study.
Flow cytometry analysis
Peripheral blood was drawn and analyzed immediately for the molecular phenotypes of neutrophils, using flow cytometry. The following antibodies were used: phycoerythrin-Texas Red (ECD)-conjugated anti-CD3, phycoerythrin-Cyanin 5 (PC5)-conjugated anti-CD15 (BD Biosciences, San Diego, CA, USA), phycoerythrin (PE)-conjugated anti-PD1, anti-T-cell immunoglobulin and mucin domain-containing protein 3 (Tim3), anti-T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains (TIGIT), anti-CD86, and anti-PDL1, fluorescein isothiocyanate (FITC)-conjugated anti-CD80 (MIH clones, e Bioscience, San Diego, CA, USA). The neutrophils were identified as CD15
+CD3
− populations [
11] and the membranous markers were detected by flow cytometry with triple staining. Briefly, 50 μL of fresh heparinized whole blood were incubated simultaneously with 5 μL ECD-conjugated anti-CD3, 5 μL PC5-conjugated anti-CD15 and 5 μL fluorescence-conjugated antibodies targeting other membranous molecules on ice in the dark for 30 minutes. Cells incubated with PE- and FITC- conjugated mouse IgG were used as isotype controls. All flow samples were analyzed with a CYTOMICS FC 500 flow cytometer (Beckman Coulter Inc., Brea, CA, USA) and associated software programs (CXP).
Autoantibodies measurement
The antinuclear antibodies (ANA) were detected using the indirect immunofluorescence method with a commercially available diagnostic kit (EUROIMMUN, Germany) according to the manufacturer’s instructions [
17]. Serum samples from SLE patients were prepared at various dilution factors as follows: 1:100, 1:320, 1:640 and 1:1000. The sample was defined as ANA-positive when the signal could be detected with the serum diluted at 1:100. Anti-double-stranded DNA (Anti-dsDNA) Abs of IgG in serum were measured by commercially available ELISA kits (Kexin, Shanghai, China). Anti-extractable nuclear antigens (ENAs) antibodies including anti-Sjogren’s syndrome-related antigen A (anti-SSA), anti-Sjogren’s syndrome-related antigen A (anti-SSB), anti-Ro52, anti-Smith (anti-Sm), anti-nRNP/Sm, anti-ribosomal ribonucleoprotein (anti-rRNP), and anti-nucleosome antibody were determined by immunoenzyme dot assay (EUROIMMUN, Germany) according to the manufacturer’s instructions. The results of anti-ENA detection were determined as negative (–) or positive (+, ++, +++) by EUROBlotOne.
Serum IgG, C-reactive protein (CRP), Complement 3 (C3) and C4 measurement
The concentrations of serum immunoglobulin G (IgG), CRP, C3, and C4 were determined by nephelometry methods according to the instructions described by the manufacturer (IMMUNE800, Beckman Coulter).
Erythrocyte sedimentation rate (ESR) and routine urine and blood measurements
ESR and routine urine and blood measurements were determined according to the instructions described by the manufacturer.
Statistical analysis
Statistical analysis and graphic presentation were carried out with GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). The t test was used when normal data distribution was confirmed; otherwise, the nonparametric Mann–Whitney test was used to analyze the data. The paired t test was performed for evaluation of changes with treatment in the group of nine patients. Likewise, the Pearson method or the nonparametric Spearman method was used for correlation analysis. A value of P <0.05 was considered statistically significant.
Discussion
Neutrophils represent the most abundant leukocyte population and traditionally recognized as one of essential effector cells of the innate immune system in humans. In recent years, an increasing interest in the role of neutrophils in interacting with and regulating the adaptive immune response has emerged [
11‐
14,
24,
25]. It is well-known that various SLE biomarkers are neutrophil-related and increased frequencies of neutrophils are found in SLE. However, the immunomodulatory roles and mechanisms of neutrophils in the onset and development of SLE are poorly understood. It is well-known that the expression of costimulatory molecules plays an important role in determining the activation status and function of immune cells. Some costimulatory molecules, especially some immunosuppressive costimulatory molecules, such as PD1, PD-L1, Tim-3 and TIGIT, have been reported to have abnormal expression on peripheral T cells, B cells, monocyte or natural killer cells in patients with SLE [
26‐
30]. In this study, for the first time, we investigated the expression of CD80, CD86, PD1, PD-L1, Tim-3, CD40 and TIGIT on neutrophils from patients with SLE, and showed that the frequency of PD-L1-expressing neutrophils was significantly increased in patients with SLE compared with healthy individuals. Moreover, our research revealed that the frequency of PD-L1-expressing neutrophils was associated with disease activity and severity of SLE.
SLE is one of systemic autoimmune diseases characterized by elevated autoimmune antibodies. In this study, the serous levels of ANA, anti-dsDNA and anti-ENA, including anti-SSA, anti-SSB, anti-Ro52, anti-Sm, anti-nRNP/Sm, anti-rRNP, and anti-nucleosome, the hallmark antibodies of SLE, were first determined and analyzed for their relationship with the frequency of PD-L1-expressing neutrophils. Data showed that the frequency of PD-L1-expressing neutrophils was significantly increased in patients with a high ANA titer and positive anti-nRNP/Sm, suggested that PD-L1-expressing neutrophils may be associated with autoimmune responses in SLE. However, although there was a tendency towards an increase in patients with positive anti-dsDNA, the frequency of PD-L1-expressing neutrophils was not significantly correlated with the anti-dsDNA titer. As the ds-DNA titer would decrease following therapy with corticosteroids and immunosuppressive drugs, the poor correlation between the frequency of PD-L1-expressing neutrophils and anti-dsDNA may be due to the fact that 87 % of SLE patients had received therapy prior to participation in the study. It is well-known that autoimmune response is a kind of chronic inflammation against self-antigens. So the correlation between the frequency of PD-L1-expressing neutrophils and inflammatory markers was analyzed. Our results showed that the frequency of PD-L1-expressing neutrophils was positively related to ESR and IgG, but inversely associated with decreased C3.
Patients with SLE have a complex array of abnormalities involving their immune system. Hematologic disorders, such as leukopenia, erythrocytopenia, anemia or thrombocytopenia, are one of the most frequent types of disorder in patients with SLE [
31‐
33] and are associated with disease activity in SLE. In this study, we observed that PD-L1 expression on neutrophils was inversely associated with anemia and erythrocytopenia, as marked by reduced HGB level, HCT and RBC count, respectively. In addition, results showed that the frequency of PD-L1-expressing neutrophils was significantly elevated in SLE patients with fever and cutaneous manifestations. Although, there was no correlation between the frequency of PD-L1-expressing neutrophils and renal involvement, the results showed that the frequency of PD-L1-expressing neutrophils was significantly elevated in SLE patients with hematuria, but not with proteinuria or pyuria. It may be due to the relatively poor sensitivity of the method used for urinary protein detection. The dry chemical method was used for proteinuria and hematuria analysis in this study. The sensitivity is 0.1 g albumin/L and 0.3 mg hemoglobin/L for proteinuria and hematuria analysis, respectively. These results suggest that the frequency of PD-L1-expressing neutrophils may be correlated with disease activity in SLE. Subsequent results from the SLEDAI classification of SLE patients confirmed our speculations. Thus, we established the correlation between the frequency of PD-L1-expressing neutrophils and disease activity in SLE.
To further investigate the clinical significance of the PD-L1-expressing neutrophils in SLE, we performed a 15-day follow-up evaluation in 10 SLE patients who received regular treatment with corticosteroids and immunosuppressive drugs. Data showed that the percentages of PD-L1-expressing neutrophils decreased in nine of these patients and were increased in one patient after 15-day treatment with corticosteroids and immunosuppressive drugs. Due to the complex characteristics of SLE, both the clinical features and response to treatment are diverse and variable in patients with SLE [
34]. Further analysis showed that the patient with increased percentages of PD-L1-expressing neutrophils had no improvement either in the SLEDAI score or clinical manifestation. Thus, these results demonstrated that the frequency of PD-L1-expressing neutrophils is correlated with disease severity in SLE.
Previously, an immunosuppressive population of neutrophils has been identified in peripheral blood of humans in particular events, such as the presence of chronic inflammation and tumors [
35,
36]. This population of neutrophils displays a remarkable ability to suppress T cell-mediated immune response by multiple mechanisms, including production of reactive oxygen species (ROS) and arginase-1 [
11,
37], and is speculated to serve as a negative feedback mechanism preventing potential tissue damage caused by excessive immune response. Considering the immunosuppressive feature of PD-L1 and the fact that the frequency of PD-L1-expressing neutrophils is associated with disease activity and severity in SLE, our research suggests that the increased frequency of PD-L1-expressing neutrophils may serve as a negative feedback mechanism, preventing potential tissue damage caused by excessive autoimmune responses in patients with SLE.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
QL participated in designing the study, performed statistical analyses and drafted the manuscript. ZKH participated in the design of the study and helped to revise the manuscript. JQY carried out flow cytometry analysis and drafted the manuscript. YTD performed statistical analyses and drafted the manuscript. XL carried out data acquisition of markers of autoimmune response and drafted the manuscript. LF performed statistical analyses of markers of autoimmune response and drafted the manuscript. YG carried out data acquisition for markers of inflammation and drafted the manuscript. HJ performed statistical analyses of markers of inflammation and drafted the manuscript. BHJ performed data acquisition of disease activity and severity, and drafted the manuscript. QSH carried out data acquisition, performed statistical analyses and drafted the manuscript. JML conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.