Pertussis toxin in involves the development of interstitial lung disease in an experimental autoimmune myositis mice modelby inducing the formation of neutrophil extracellular traps / 中华风湿病学杂志

Objective:To test the hypothesis that Pertussis toxin (PTX) can promote the occurrence of interstitial lung disease (ILD) in experimental autoimmune myositis (EAM) model and clarify the potential pathogenic mechanism.Methods:EAM mice model were induced by Skeletal muscle thomogenate with or without PTX, and the relationship between ILD phenotypes and neutrophil extracellular traps (NETs) infiltration was analyzed by histopathological and serological studies in EAM with PTX group and EAM without PTX group. Healthy mice were given PTX alone intraperitoneally to clarify whether NETs formation could be induced in vivo, and neutrophils separated from healthy human blood were intervened with PTX to induce NETs formation in vitro. The data was tested for normality using Shapiro-Wilk. Statistical methods and were analyzed using t-test or ANOVA, and multiple comparisons between different groups were tested using Tukey test. Results:Compared with EAM without PTX group, lung tissues in EAM with PTX group had multiple pathological changes similar to polymyositis/dermatomyositis-related ILD. Nonspecific interstitial pneumonia and usual interstitial pneumonia were the main pathological types. The pulmonary interstitial lesions were accompanied by significant infiltration of NETs; and serum NETs markers levels were obviously elevated in EAM with PTX group, compared with the control group [ n=5, (87±10) ng/ml], cfDNA levels were statistically significantly elevated in both the EAM without PTX group [ n=4, (115±27) ng/ml] and the EAM with PTX group [ n=7, (150±50) ng/ml] ( F=4.24, P=0.038); Cit-H3-DNA levels were elevated in the EAM without PTX group ( n=4, 0.24±0.09), and in the EAM EAM with PTX group ( n=6, 0.33±0.11) compared with the control group ( n=4, 0.13±0.02) ( F=6.21, P=0.016). After PTX intervention, serum cfDNA levels were higher in the PTX group [ n=3, (100±40) ng/ml] than in the control group [ n=3, (45±12) ng/ml, t=2.27, P=0.086]; PTX also induced neutrophils to form NETs in vitro. Conclusion:PTX may promote the development of ILD in EAM mice model by inducing the formation of NETs, indicating that EAM mice can serve as a model for targeting NETs to study the pathogenesis ILD.

A preliminary study on the pathogenesis of neutrophil extracellular traps in rheumatoid arthritis / 中华风湿病学杂志

Objective To explore the potential effects of neutrophil extracellular traps (NETs) on rheumatoid arthritis synovial fibroblasts (RA-FLSs).Methods The synovial tissues of RA patients were isolated and cultured in vitro.Peripheral blood neutrophils were extracted from healthy volunteers and used to stimulate NETs' formation,following with NETs' extraction.MTS proliferation assay was used to evaluate the effect of NETs on the proliferation of RA-FLSs.QRT-polymerase chain reaction (q-PCR) was used to determine the expression of connective tissue growth factor (CTGF) mRNA in cells treated with NETs-stimulated RA-FLSs for 60 h.The results were processed using paired sample t-test and one-way analysis of variance (ANOVA).Results The isolated and purified neutrophils could form NETs by in vitro stimulation.The concentration of extracted NETs-DNA was 58.5 ng/μl (1×106 cells).Compared with the control group (0 μl NETs),NETs could promote the proliferation of RA-FLSs.With the increase of NETs' concentration,the proliferation of RA-FLSs was also enhanced (F=99.519,P＜0.05).Compared with the control group (0 μl NETs),10 μl NETs could significandy promote the proliferation of RA-FLSs (t=-12.226,P＜0.01).Pretreatment of NETs with DNase Ⅰ inhibited its effect on promoting the proliferation of RA-FLSs (t=-2.376,P=0.049),NETs stimulated the upre-gulation of CTGF mRNA expression in RA-FLSs [(30.7±0.5),t=12.13,P＜0.01].Conclusion NETs can promote the proliferation of RA-FLSs and stimulate the up-regulation of CTGF mRNA in RA-FLSs in vitro.

Correlation research of low density granulocytes with interstitial lung disease in dermatomyositis / 中华风湿病学杂志

Objective This study aims to test the hypothesis that low density granulocytes (LDGs) is involved in the pathogenesis of DM associated-Interstitial lung disease (ILD).Methods Forty eight DM patients (28 with ILD) and 19 age-and sex-matched healthy Chinese volunteers were recruited to this study.LDGs percentage in peripheral blood mononuclear cells (PBMCs) was tested by flow cytometry.Neutrophilrelated genes (LL-37,MPO and MMP-8) expressions in PBMCs were tested by quantitative RT-PCR.Myositis disease activity assessment visual analogue scales (MYOACT) was used to assess the disease activity.Percentages of LDGs were compared in patients with ILD and without by using unpaired t test with Welch's correction,the correlations between LDGs and clinical parameters were further analyzed by linear correlation analysis.The expressions of neutrophil-related mRNA and proteins in PBMCs were compared by using MannWhitney U test.Results LDGs percentage in PBMCs was 7.1-fold higher in DM patients than healthy controls [(9.1±11.5)％ vs (1.3±0.7)％,t=4.664,P＜0.01].LDGs percentage in PBMCs was 2.7-fold higher in DM patients with ILD than DM patients without ILD [(12.3±14.1)％ vs (4.5±2.6)％,t=2.835,P=0.008 3].The mRNA expression level of LL-37,MPO and MMP-8 and LL-37 protein levels in the DM group were significantly higher than those in the control group.LDGs percentage positively correlated with MYOACT lung disease activity scores (r=0.439,P=0.010).Conclusion Percentage of LDGs in PBMCs is significantly increased in DM patients with ILD and positively correlated with MYOACT lung disease activity scores,suggesting that abnormall increasing of LDGs is a potential contributor to the pathogenesis of DM-associated ILD.

The research of myosin-binding protein C in duced autoimmune myositis model / 中华风湿病学杂志

Objective To establish a new murine model of experimental autoimmune myositis by immunizing with MYBPC2 protein. Methods The purified Myosin-binding protein C, fast type (MYBPC2) was emulsified with complete Freundˊs adjuvant, then C57BL/6 mice were immunized by multi-point subcutaneous injection (0, 7 days), and intraperitoneal injection of pertussis toxin 2 μg simultaneously. The pathological changes of mice with different immunizing dose at the preconceived time were ex-plored. Mean-while, mice were immunized with 600 μg each time, and the muscle endurance was tested on the 21th day. The expression of major histocompatibility complex (MHC) class-Ⅰ and the surface biomarkers of the inflammatory cells in muscle tissues were observed. Mann-Whitney U test was used for statistical analysis. Results ① With the increase of immunizing dosage, muscle damage and inflammation tended to be more serious. On the 21th and 28th day, muscle lesions were most significant. Muscle fiber degeneration and necrosis and inflammatory cell infiltration could be seen in the experimental group. ② Compared with the control group, muscle endurance of mice in the experimental group decreased significantly [(6.1 ±1.3) min versus (9.2±1.6) min, U=2.00, P=0.017]. The MHC class-Ⅰ on the muscle fiber surface of the experimental group was positive, scattered infiltration of CD4 +, CD8+ T ly-mphocytes and CD68 + macrophages between muscle fibers and around the vascular areas could be observed, and CD20+B lymphocytes mainly distributed in the area around the blood vessels, nevertheless rarely seen between muscle fibers. Conclusion Exper-imental autoimmune myositis models of mice have been successfully induced by immunizing with MYBPC2 in China for the first time, and similar clinical and pathological features of human polymyositis could be observed. This new model can be used for studying the pathogenesis of autoimmune myositis.

Elevated plasma level of circulating cell-free DNA may derive from NETosis and is associated with active lupus nephritis in systemic lupus erythematosus / 中华风湿病学杂志

Objective To explore the correlations between elevated cfDNA with lupus nephritis and indentify the influencing factors of cfDNA in systemic lupus erythematosus (SLE).Methods Fifty four patients with SLE [37 patients with lupus nephritis (LN) and 43 age-and sex-matched healthy controls] were included in the study.In 37 LN patients,26 patients were at active stage,and 11 patients were in remission.cfDNA concentration was measured with Picogreen Kit and low-density granulocytes (LDGs) was tested by flowcytometer.Correlation and regression analysis were performed to discover whether cfDNA is related to LN and identify the influencing factor of cffDNA.Results The cfDNA in SLE group was (237±40) ng/ml,which was significantly higher than that in healthy control group (188±41 ng/ml,P＜0.01).cfDNA in LN group was significantly higher than that in patients without LN (NLN) (247±47 ng/ml vs 214±31 ng/ml,P=0.028).cfDNA in patients with active LN was significantly higher than that in patient with inactive LN (RLN) (254±50 ng/ml vs 216±29 ng/ml,P=0.035).In SLE group,cfDNA was positively correlated with quantitative 24-hour urinary protein (r=0.350,P=0.013) and reversely correlated with albumin (r=-0.500,P＜0.01) and endogenous creatinine clearance rate (Ccr) (r=-0.354,P=0.044).Percentage of LDGs in peripheral blood mononuclear ceils (PBMCs) of the SLE group was (8.3± 12.9)％,significantly was higher than that in healthy controls [(1.2±0.7)％,P=0.004].The cfDNA was positively correlated with LDGs (r=0.636,P=0.002) and neutrophils (r=0.599,P＜0.01).Conclusion NETs excessively released by neutrophils as well as LDGs may be one of the main reasons for elevated cfDNA level in SLE.cfDNA level is associated with LN activity,suggesting that there is a intrinsic link between NETs-related biomarkers and active LN and that more specific biomarkers of NETs may become a clinical biomarker for active LN.

Study of association between neutrophil extracellular trap and interstitial lung disease in dermatomyositis patients / 中华风湿病学杂志

Objective This study was focused on the association between neutrophil extracellular traps (NETs) and interstitial lung disease (ILD) in patients with dermatomyositis (DM).Methods Thirty six patients who satisfied the Bohan & Peter criteria for DM were recruited to this study,among whom 19 were complicated with ILD.Forty seven age and sex matched healthy Chinese volunteers were selected to be control subjects.The plasma samples of these patients were tested for the formation and degradation of NETs.Results DM plasma induced more NETs formation than control plasma did [(246±93) RFUs vs (192±53) RFUs,P=0.002].Compared to control,DM plasma exhibited a signficantry decreased ability to degrade NETs.Further mere,compared with DM patients without ILD (DMNL),DM patients with ILD (DML) could not degrade NETs completely [(83±13)％ vs (59±21)％,P＜0.01].All four DM patients with subacute ILD exhibited a significantly lower ability to degrade NETs than patients with chronic or asymptomatic ILD [(36±14)％ vs (65±19)％,P=0.0139].Conclusion These data show that more NETs formation is induced by plasma and DML fails to completely degrade NETs.These suggest that NETs may play a role in the pathogenesis of DM and DM-associated ILD.