Transcriptome Sequencing And Cytokines Of Peripheral Blood Mononuclear Cells Stimulated By LPS In Patients With Behcet’s Disease-associated Uveitis

PurposeTo explore differential genes Changes in the expression,function and key biological information pathways of BU to reveal the pathogenesis of BU and provide new possible targets for its treatment.Bacterial lipopolysaccharide（LPS）stimulated differences in peripheral blood mononuclear cells（PBMCs）transcriptome sequencing and cytokines in patients with active Behcet disease-associated uveitis（BU）,patients in remission,and normal subjects were analyzed.MethodsTen active BU patients and seven BU remission patients who were treated in Lanzhou University First Hospital from September 2018 to May 2019 were selected,and ten healthy volunteers were selected as controls.12 ml of venous blood was drawn from each person and PBMCs were isolated.The isolated PBMCs were identified by immunofluorescence.The isolated PBMCs were implanted into 12-well plates and 24-well plates respectively,and LPS(1ug·m L^-1 for both)was added to stimulate culture.RNA of PBMCs in 12-well plates was extracted 12 h after LPS stimulation,and 4 cases of RNA in each group were selected for RIN>7,28S:18S>1.8,and the initial RNA content was between 0.1～1ug were sequenced by Illumina Hiseq2500 transcriptome,and key differentially expressed genes were screened by gene functional enrichment（GO）,KEGG enrichment signal pathway,gene co-expression network and protein interaction network.Cell supernatant was collected from the24-well plate at 0 h,3 h,6 h,12 h and 24 h after LPS stimulation.The concentrations of IL-1,IL-6,TNF-αand IL-10 were determined by enzyme-linked immunosorbent assay（ELISA）.Results（1）Compared with PBMCs transcriptome sequencing between BU active and normal subjects,found 1226 differentially expressed genes,including 737up-regulated genes and 489 down-regulated genes.Compared with PBMCs transcriptome sequencing between BU active and remission stages,1042 differentially expressed genes were found,including 517 up-regulated genes and 525down-regulated genes.GO analysis shows that significantly enriched GO Terms mainly focused on the reaction to lipopolysaccharide,various immune responses,platelet activation,hemostasis and coagulation process,and the activities of cytokines and chemokines.The significant signaling pathways of differential genes mainly include natural killer cell mediated cytotoxicity,Fc-gamma receptor signaling pathway,chemokine and cytokine-mediated signaling pathway,JAK-STAT signaling pathway,NF-κB signaling pathway,etc.（2）Eight important genes related to BU were screened by analyzing differential gene co-expression and protein interaction network.PLAU and PLAUR on the NF-κB signaling pathway were up-regulated in the comparison of BU active phase and normal human transcriptome sequencing,and down-regulated in the comparison of BU active phase and remission phase transcriptome sequencing.（3）The results of ELISA showed that PBMCs in active BU and remission stages had transient low response to LPS stimulation.Conclusion（1）The pathogenesis of BU is related to Toll-like receptor signaling pathway and multiple inflammatory and immune pathways,which were found by GO and KEGG enrichment analysis;（2）A number of important genes related to the pathogenesis of BU were screened out,and new possible targets for the treatment of BU were provided through the analysis of differential gene co-expression and protein interaction network.The role of PLAU in BU deserves further study;（3）The transient hyporesponsiveness of PBMCs in patients with active and remission BU stimulated by LPS may be related to endotoxin tolerance.