Expression Profiles In Circulating Neutrophils From Patients With ARDS And Human Pulmonary Microvascular Endothelial Cells Exposed To Lipopolysaccharide

1.BackgroundThe acute respiratory distress syndrome(ARDS)is characterized by diffuse damage of alveolar-capillary barrier,immune cell infiltration,protein-rich edema fluid in alveoli and severe gas-exchange abnormalities.Despite over 50 years of clinical and basic studies,ARDS is still a critical challenge with high mortality worldwide.ARDS increases healthcare costs and impairs quality of life seriously.Therefore,it is urgent and highly demanded for getting a better understanding of the pathogenesis of ARDS.Polymorphonuclear neutrophils(PMNs)are crucial for controlling infections as innate immune system cells.Circulating PMNs become activated and penetrate the alveolar-capillary barrier into the airspaces in the progression of ARDS.PMNs in the alveoli inflammatory microenvironment become further activated to play an important role in phagocytosing pathogens,releasing reactive oxygen species and inducing neutrophil extracellular traps.Subsequently,activated neutrophils lead to alveolar damage and further loss of lung function.However,the mechanisms of blood PMNs activation and infiltration in the development of ARDS remain poorly understood.The rapid development of high-throughput detection technology in recent decades has provided technical support for genome-wide analyses in the progression of lung disease,such as ARDS and asthma.However,one of the major issues in previous high-throughput studies of ARDS was that samples were whole blood or total leukocytes rather than purified neutrophils.In the present study,we identified differential expressed genes(DEGs)in blood PMNs from patients with ARDS compared with healthy volunteer(HVT)blood PMNs in GSE76293.To identify the ARDS-specific blood neutrophil phenotype,we also identified DEGs in PMNs exposed to plasma from severe septic patients compared to uninfected controls in GSE49757.Using integrated bioinformatics methods,we predicted key pathways and hub genes involved in the ARDS-specific circulating neutrophil phenotype.Our resu lts may provide novel therapeutic targets or biomarkers for ARDS.2.ObjectiveThe aim of the present study was to identify key pathways and genes ARDS-specific blood neutrophil phenotype and rovide novel therapeutic targets or biomarkers for ARDS.3.Methods3.1 Microarray dataTo investigate the ARDS-specific neutrophil phenotype,we searched expression profile of ARDS blood PMNs and chose the dataset GSE76293 and GSE49757 from the GEO(Gene Expression Omnibus)data repository.In the current study,12 ARDS blood PMNs samples in parallel with 12 HVT blood PMNs samples in GSE76293 and 20 PMNs samples stimulated with severe sepsis plasma compared with 19 PMNs samples stimulated with HVT plasma in GSE49757 were used for analysis.3.2 Identification of DEGsDEGs were identified using GE02R(http://www.ncbi.nlm.nih.gov/geo/geo2r/),which is an online tool based on the GEOquery and Limma R packages.The genes that met the cut-off criteria of an adjusted p value<0.01 and a |log2 fold change|>1 were considered DEGs.To indicate the intersection among DEGs between GSE49757 and GSE76293,Venn diagram was produced by a venn webtool(http://bioinformatics.psb.ugent.be/webtools/Venn/).3.3 Gene ontology(GO)and pathway enrichment analysesDAVID was used to analyze the gene ontology and pathway of DEGs.P value<0.05 was used as thresholds to define significantly enriched terms of GO or pathways.3.4 Protein-protein interactions(PPI)network and module analysis Search Tool for the Retrieval of Interacting Genes(STRING;http://string-db.org,version 10.5)online database was used to predict interactions of DEGs.PPI network was drawn by Cytoscape(version 3.7.1).Furthermore,the plugins CytoNCA and MCODE of Cytoscape were used to identify the hub genes and modules in the PPI network.3.5 Transcription factor(TF)regulatory network analysisThe iRegulon plugin in Cytoscape was used to predict TFs of the selected hub DEGs.A normalized enrichment score(NES)>4 was considered the threshold value.3.6 Relative mRNA expression level of hub genesTo analyze the relative mRNA expression level of seven hub genes in GSE49757 and GSE76293,we downloaded the matrix data of the two datasets from the GEO data repository and analyzed the log2 normalized signal intensity of selected hub genes using GraphPad Prism 7.04.3.8 Statistical AnalysisAll statistical analyses in this study were performed using GraphPad Prism 7.04(GraphPad Software,San Diego,CA,USA),and p<0.05 was considered to be significant.Data are presented as mean ± SEM.Student's t-test was used to compare difference.4.Results4.1 Identification of DEGsIn total,1120 mRNAs were significantly differentially expressed in ARDS blood PMNs compared with HVT blood PMNs.There were 971 DEGs in PMNs exposed to severe septic plasma compared with uninfected controls.A total of 220 DEGs overlapped between GSE49757 and GSE76293 in the Venn diagram.4.2 GO and pathway enrichment analysesDEGs in GSE76293 were mainly associated with the following biological processes:apoptotic process,response to oxidative stress,response to lipopolysaccharide,response to tumor necrosis factor,leukotriene signaling pathway.The results also indicated that DEGs in GSE76293 were mainly enriched in the following pathways:MAPK signaling pathway,FoxO signaling pathway,AMPK signaling pathway,TNF signaling pathway.DEGs in GSE49757 were mainly associated with the following biological processes:inflammatory response,response to lipopolysaccharide,apoptotic process,immune response,positive regulation of cytokine production,positive regulation of NF-kappaB signaling.The results also indicated that DEGs in GSE49757 were mainly enriched in the following pathways:NF-kappa B signaling pathway,Cytokine-cytokine receptor interaction,NOD-like receptor signaling pathway,TNF signaling pathway.4.3 PPI network analysisThe PPI network of 899 nodes with 3757 protein interaction pairs was constructed to identify hub genes in ARDS circulating PMN activation in GSE76293.We identified 30 hub genes and three most significant modules in the PPI network.GAPDH,AKT1,MAPK14,MAPK8,IL8,PIK3CB and MMP9 were all in the three most significant modules.4.4 TF regulatory network analysisThe TFs of the top 50 hub genes in the PPI network were predicted.With a threshold of an NES>4,a total of six transcription factors(E2F1,NFKB1,NFYA,PBX3,EGR1,RELA)were revealed to be associated with 30 target hub genes.4.5 Relative mRNA expression level of hub genesWe found that AKT1 and IL8 were downregulated,while MAPK14 was upregulated in both GSE49757 and GSE76293.At the same time,the relative expression trends of GAPDH,MAPK8,PIK3CB and MMP9 were different between GSE49757 and GSE76293.5.ConclusionIn conclusion,we identified key pathways and genes involved in the ARDS-specific circulating neutrophil phenotype.Several hub genes,such as MAPK8,PIK3CB and MMP9,may play important roles in ARDS-specific blood neutrophil activation and represent promising targets for the diagnosis and treatment of ARDS in the future.