Correlation And Mechanism Research Of Abnormal Regulation Of NETs With Interstitial Lung Disease In Dermatomyositis And Polymyositis

BackgroundInterstitial lung disease (ILD) is the most common complication in Dermatomyositis (DM) and polymyosits (PM). Although ILDs are closely related to bad prognosis, its pathogeneses remain unclear. New researches indicted that low density granulocytes (LDGs) can release proinflammatory cytokines, injure vascular endothelial and form neutrophil extracellular traps (NETs). Abnormal regulation of NETs is reputed to play an important role in the pathogenesis of autoimmune diseases and inflammatory lung diseases. However, whether the percentage of LDGs and the regulation of NETs are normal and how NETs contribute to the pathogenesis of ILD in DM/PM are unknown.ObjectiveThe first aim of this study is to investigate whether LDGs is involved in pathogenesis of ILD in DM and to analyze the correlations between LDGs with clinical items.The second aim of this study is to test the hypothesis that NETs may be pathogenic in DM/PM and to explore the possible mechanisms.MethodsStudy Ⅰ. Forty eight DM patients were recruited for this study, among whom28complicated with ILD. Nineteen age-and sex-matched healthy Chinese volunteers were selected to be control subjects. LDGs percentage in peripheral blood mononuclear cells (PBMCs) was tested by flow cytometer. Neutrophil-related protein expression in PBMCs was tested by enzyme-linked immunosorbent assay (ELISA). Neutrophil-related gene expressions in PBMCs were tested by quantitative RT-PCR. Myositis Disease Activity Assessment Visual Analogue Scales (MYOACT) was used to assess the disease activity. Comparisons of LDGs and others items were performed between DM patients with ILD and without. Correlations between LDGs with other clinical items were performed with linear correlation analysis. Study Ⅱ. Plasma samples from97DM/PM patients (72DM,25PM) and54healthy controls were tested for the capacities to induce and degrade NETs. Plasma DNase I activity was tested by the radial enzyme-diffusion method to further explore possible reasons for the incomplete degradation of NETs. Results from35DM patients and seven PM patients with ILD were compared with results from DM/PM patients without ILD.ResultsStudy Ⅰ. Compared with healthy control (1.28%±0.73%of total PBMCs), DM patients showed significantly higher percentage of LDGs (9.06%±11.50%of total PBMCs, P<0.0001). Compared with DM patients without ILD, DM patients with ILD exhibited significantly higher percentage of LDGs in PBMCs (4.54%±2.61%vs.12.29%±14.13%, P=0.0083). The mRNA expression level of LL-37, MPO and MMP-8and LL-37protein levle in DM group were significantly higher than that in Control group. The percentage of LDGs positively correlated with MYOACT lung disease activity scores, CKMB, tumor markers, neutrophil count, TG and HDL, and inversely correlated with tT3and fT3.Study Ⅱ. Compared with control subjects, DM/PM patients exhibited a significantly enhanced capacity for inducing NETs (191.6±52.88RFUs vs.246±93.48RFUs, P=0.002), which was supported by elevated levels of plasma LL-37and circulating cell-free DNA (cfDNA) in DM/PM. NETs degradation in DM/PM paients was significantly lower than in control patients. Compared with DM/PM patients without ILD, DM/PM patients with ILD degraded less NETs in vitro (83.41%±12.64%vs.58.58%±21.4%, P=0.0002); Plasma DNase I activity in PM and DM were0.1822±0.0940U/ml and0.2094±0.1112U/ml, repectively, significantly lower than in control subjects (0.3933±0.1523U/ml, P<0.0001). Compared with DM/PM patients without ILD, DM/PM patients with ILD exhibited a lower DNase I activity (0.2301±0.1187U/ml vs.0.1677±0.077U/ml, P=0.0039). Patients with anti-Jo-1antibodies and patients with subacute ILD exhibited a significantly decreased DNase I activity. Glucocorticoid therapy seems to improve DNase I activity.ConclusionDM patients, especially patients with ILD, exhibited a significantly increased percentage of LDGs in PBMCs. Moveover, the percentage of LDGs positively correlated with MYOACT lung disease activity scores, suggesting that LDGs may play a role in pathogenesis of DM-associated ILD. The significantly increased mRNA expression level and protein level of neutrophils-related markers in PBMCs indicate that LDGs are another group of cells that can potently forme NETs and LDGs may contribute to ILD in DM patients by excessively forming NETs.The excessively formed NETs cannot be completely degraded because of decreased DNase I activity in DM/PM patients, especially in patients with ILD, which makes DM/PM patients expose to large amounts of NETs. Moreover, abnormally increased LDGs may contribute to abnormal regulation of NETs. All of which may cause constant and severe damage of lung and further induce ILD. It is reasonable to suppose that abnormal regulation of NETs may be involved in the pathogenesis of DM/PM and could be one of factors that initiate and aggravate ILD.