| Objectives:To investigate the association between neutrophil and clinical characteristics of patients with DM-ILD,and to analyze the clinical risk factors related to the DM-ILD.To screen differentially expressed genes(DEGs)in transcriptional level between patients with dermatomyositis associated interstitial lung disease(DM-ILD)and healthy controls.To verify the expression level of PLAUR in peripheral blood of DM-ILD patients,and explore the effect of PLAUR on neutrophil activation,apoptosis and autophagy in neutrophil-like cells.This study would provide potential biomarkers and prognosis indexes for diagnosis and therapy of DM-ILD,.Methods:The correlation between the level of neutrophil and clinical characteristics was analyzed.A cross-sectional study of DM-ILD was conducted to explore the clinical risk factors of DM in the second affiliated Hospital of Kunming Medical University from January 2016 to December 2018.Peripheral blood samples from 6 DM-ILD patients and 6 healthy controls were collected.Next generation sequencing technology screened DEGs in PBMC.Analysis of DEGs enrichment by Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway,Disease Ontology(DO).The python toolkit rMATs analyzed alternative splicing(AS)event at transcriptome sequencing data.DM-ILD associated immune cells was analyzed by CIBERSORT.Plasminogen activator and urokinase receptor(PLAUR),the most important genes related to DM-ILD were screened,and the expression of PLAUR was positively correlated with the abundance of neutrophils.The relative expression level of PLAUR in DM-ILD group and healthy control group was detected by real-time quantitative polymerase chain reaction(RT-qPCR).Then human acute myeloid leukemia cell lines with high expression of PLAUR were screened,and HL60 cells induced by all-trans retinoic acid(ATRA)to construct a neutrophil-like model.The cells were divided into three groups:control group(no transfection),empty load group(transfection empty load)and siRNA group(transfection siRNA).The cultured cells were starved and stimulated by IL-17.The expression levels of CD11b,neutrophil extracellular trapping network(NETs)and myeloperoxidase(MPO)were detected by flow cytometry(FCM)and enzyme-linked immunosorbent assay(ELISA),respectively.The expression of apoptosis and autophagy were detected by FCM,WB,immunofluorescence and transmission electron microscope,respectively.Results:A total of 133 DM patients were enrolled in the group from January 2016 to December 2018.Spearman correlation analysis showed that neutrophils were positively correlated with MDAAT,ESR,CRP,Fib,LDH,r values were 0.240,0.372,0.248,0.218,0.177,respectively,and P values were less than 0.05.The ROC curve revealed that the sensitivity and specificity of absolute neutrophil value in assessing the occurrence of DM-ILD were 0.604 and 0.637,respectively,when the cut-off value was 4.575.And the area under the curve(AUC)was 0.648,95%CI(0.552～0.743).Cross-sectional analysis showed that the prevalence rate of DM-ILD was 39.8%,95%confidence interval(26.6%～53.0%).Univariate logistic analysis showed that there were differences in MDAAT score,absolute value of neutrophils,Fib,ESR and CRP levels.Further analysis of multivariate Logistic regression showed that the independent factors related to ILD were MDAAT score(OR=2.681,95%CI 1.627-4.415,P=0.000)and smoking(OR=130.194,95%CI 2.738-6191.031,P=0.013).A total of 2018 DEGs were identified between DM-ILD and healthy controls.GO and KEGG enrichment analysis showed that DEGs was mainly involved in the biological processes and pathways related to immunity and inflammation.DO enrichment identified 35 candidate key genes involved in skin and lung diseases.At the same time,a total of 886 differentially expressed AS events were found between DM-ILD and healthy volunteers,and 697 genes were involved.After overlapping DEGs and differential AS genes,the only differential gene PLAUR involved in immune-related biological processes and complement and coagulation cascade was screened.Immune infiltration analysis showed that compared with the healthy control group,the number of neutrophils was significantly increased in DM-ILD group.There was a positive correlation between PLAUR expression and neutrophil abundance.RTqPCR detection showed that the relative expression of PLAUR in DM-ILD patients was significantly higher than that in healthy controls.Flow cytometry and ELISA results showed that CD11b in siRNA group decreased significantly,and the levels of NETs and MPO in siRNA group were significantly lower than the other groups(P<0.05).Flow Annexin V/PI detection of apoptosis showed that the expression of apoptosis was increased in siRNA group.The expression of caspase3 and bax proteins increased and the expression of bcl-2 decreased in siRNA group were detected by WB.In addition,immunofluorescence microscope showed that the expression of LC3 positive granules in the siRNA group was significantly higher than that in the control group;transmission electron microscope showed that the number of autophagosomes and mitochondria was more in the control group.In the knockout PLAUR group,more double-layer membrane autophagosomes could be seen and the number of mitochondria decreased.Conclusions:In this study,a clinical correlation analysis showed that neutrophils were positively correlated with MDAAT score,inflammatory indicators CRP and ESR,coagulation index Fib,and LDH.Absolute neutrophil values can be used for the evaluation of DM-ILD.Through cross-sectional analysis,when DM patients have a history of smoking and MDAAT high score,attention should be paid to screening potential ILD,strengthening lung disease monitoring.The disease-related gene PLAUR was found by transcriptome sequencing and systematic analysis of AS events in DM-ILD.Comprehensive bioinformatics analysis confirmed that neutrophils play an important role in DM-ILD.In DM-ILD,the expression of PLAUR increased.By constructing a neutrophil-like model with knockout PLAUR,it was found that PLAUR promoted neutrophil activation and inhibited its apoptosis and autophagy.It means that PLAUR may promote the activation of neutrophils in the lung,limit neutrophils clearance,and eventually lead to neutrophils associated lung injury.These findings enrich our understanding of DM-ILD and may be helpful for the diagnosis and treatment of DM-ILD in the future. |
