| ObjectiveSystemic lupus erythematosus (SLE) is an autoimmune disorder characterized by multiple autoantibodies against self-directed antigens and multisystemic involvement. It has become evident that dysfunction of immune regulation mechanisms, such as imbalance of pro-inflammatory cytokines and anti-inflammatory cytokines, contributes to the development of SLE. Interleukin-37(IL-37) is recently identified as a natural suppressor of innate inflammatory and immune responses, belonging to IL-1cytokine family. Constitutive IL-1F7mRNA is found in the testis, thymus and uterus, and can be induced in peripheral blood mononuclear cells (PBMCs) and dendritic cells. IL-37is not constitutively expressed in tissues from healthy subjects, but high in inflammatory tissues. Glucocorticoid (GC) remains the cornerstone of the treatment of systemic lupus erythematosus (SLE), despite advances in immunosuppressive drugs, therapeutic protocols and development of new drugs. Previous studies had showed that glucocorticoid inhibited the expression and action of most cytokines. However, the effect of glucocorticoid against IL-37remains unclear. In our present study, serum levels as well as mRNA expression in peripheral blood mononuclear cells (PBMCs) of IL-37, IL-18, IL-18BP, IL-6, IFN-γ and TGF-β were tested to investigate the possible role of IL-37in SLE and the possible effects of glucocorticoid on IL-37and other cytokines activities in SLE patients.Patients and Methods PatientsTwenty newly diagnosed severe SLE patients with systemic lupus erythematosus disease activity index (SLEDAI)>10were enrolled into the present study and received prednisone1mg/kg/day for fourteen consecutive days. None of them had been treated with GCs and other immunosuppressive drugs prior to first sampling. Peripheral blood samples were obtained from all patients prior to and two weeks after glucocorticoid therapy. Twenty healthy adult volunteers were enrolled as control group.Isolation of RNA from PBMCSera obtained from all subjects by centrifugation of peripheral blood samples were stored at-80℃until detection of cytokines. Mononuclear cells were isolated from heparinzed blood by gradient centrifugation. Total RNA was isolated by Trizol. The amount of RNA was determined using the Eppendorf Biophotometer and normalized to1μg/mL for each subsequent real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) process.Cytokines detection by ELISASera cytokine levels were measured using ELISA kits for IL-37, IL-18, IL-18BP, IFN-γ, IL-6and TGF-β.Quantitative real-time reverse transcription-polymerase chain reaction analysisMultiplex real-time RT-PCR was performed for IL-37, IL-18, IL-18BP, IFN-γ, IL-6, TGF-β and the endogenous control (ACTB). Each sample was run in triplicate. The PCR products were run in an agarose gel and were confined to a single band of the expected size in all cases. A melting-curve analysis was also performed to ensure specificity of the products. We used the comparative Ct method for relative quantification of cytokine mRNA according to relative expression software tool. The amplification efficiency between the target and the reference control were compared in order to use the delta delta Ct (ΔΔCt) calculation.ResultsClinical characteristicsThere were17females and3males, aged between13and56(29.2±9.2). The course of disease from the presence of symptoms and signs to the enrollment varied from1to36months (9.5±9.6). The SLEDAI scores ranged from14.7±4.0of pre-treatment to12.8±3.1of post-treatment.Sera cytokines levels changes in SLE patientsThe plasma levels of all the cytokines including IL-37, IL-18, IL-18BP, IFN-y, IL-6and TGF-β in SLE patients with SLEDAI>10were increased significantly compared with the normal controls (p<0.05). There were no significant differences in plasma levels of IL-18, IL-18BP, IFN-y, IL-6between post-treatment SLE patients and controls. Statistically significant IL-37(p<0.05) and TGF-P (p<0.05) levels were found between post-treatment SLE patients and controls.Comparison of mRNA expressions in pre-treatment SLE patients and controlsUsing the REST software, the data are presented as the fold change in gene expression normalized to an endogenous reference gene and relative to healthy controls. The relative amount of IL-37mRNA was increased2.2-fold in pre-treatment SLE patients compared to controls (p<0.01). The expression of IL-18BP was unchanged in pre-treatment patients compared to the controls. IFN-y was up-regulated in pre-treatment SLE patients by1.8-fold compared to controls (p<0.01). IL-18, IL-6and TGF-β mRNA expressions were significantly higher in pre-treatment SLE patients by2.0-fold,2.5-fold and2.8-fold respectively compared to those in control group (p<0.01). No decreases were observed for all of the six kinds of cytokines gene expressions in pre-treatment SLE patients compared to controls. Of all the subjects, there was no significant difference for IL-18, IL-18BP, IFN-y and IL-6between post-treatment SLE patients and the control group.Correlations of IL-37with other cytokines in SLE patientsOur present study showed that plasma concentrations of IL-37correlated with IL-18(r=0.983, p<0.01), IL-18BP (r=0.985, p<0.01), IFN-γ (r=0.968, p<0.01), IL-6(r=0.980, p<0.01) and TGF-β (r=0.969, p<0.01) in pre-treatment SLE patients (Fig1). After fourteen consecutive days treatment of prednisone, plasma concentrations of IL-37correlated with IL-18(r=0.826, p<0.01), IL-18BP (r=0.833, p<0.01), IFN-γ (r=0.896, p<0.01), IL-6(r=0.853, p<0.01) and TGF-β (r =0.944, p<0.01) in post-treatment SLE patients (Fig2). There were significant correlations between IL-37level and SLEDAI score both in pre-treatment SLE patients (r=0.875, p<0.01) and post-treatment ones (r=0.577, p<0.01) in our present study.Effect of glucocorticoid on cytokines sera levels and mRNA expressions in SLE patientsThere were significant differences in plasma levels of IL-37(p=0.048), IFN-γ (p=0.013) and TGF-β (p=0.028) between pre-treatment SLE patients and post-treatment ones. No significant difference in plasma levels of IL-18, IL-18BP, and IL-6between pre-treatment SLE patients and post-treatment ones were found.The relative amount of IL-37mRNA was decreased40%in post-treatment SLE patients compared to pre-treatment ones (p<0.01). Statistically significant differences for IL-18, IL-6, IFN-γ and TGF-β mRNA expressions were also found in pre-treatment SLE patients compared with those post-treatment SLE patients.ConclusionsIn conclusion, the present data demonstrate that IL-37is not constitutively expressed in health subjects and IL-37is up-regulated in active SLE patients. The imbalance of proinflammatory and anti-inflammatory cytokines contribute to the pathogenesis of SLE. Increasing the expression of IL-37or replenishing the exogenous IL-37might be a potential therapeutic strategy for SLE patients.
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