The Study Of Transcriptomics And Inflammatory Factors In Generalized Pustular Psoriasis

Background and Objective:Generalized pustular psoriasis is a rare inflammatory disease which can relapse periodically.The clinical manifestation is the presence of acute or subacute sterile pustules on the flush red patch all over the body.GPP can affect multiple body systems and can be life-threatening.GPP generally occurs in middle-aged adults and occasionally in children.To date,the pathogenesis of GPP remains elusive,and there is no consensus for therapy.Thus,it is imperative to explore GPP pathogenesis further.To this end,we performed systematic studies on the kinetics of gene expression and alterations of inflammatory cytokine levels in the peripheral blood from GPP patients at different disease stages.We aimed to identify signaling pathways and genes that play essential roles in GPP,and thus provide insights into novel therapeutic strategies.MethodsFive patients were diagnosed with GPP at their initial visit and received six-month treatment with acitretin.Transcriptome profiling was performed by RNA-Seq with PBMC samples collected from those five patients at three time points:TO(the acute phase of GPP,prior to initiation of acitretin treatment),T1(2 weeks after treatment)and T2(6 months after treatment).Differentially expressed coding genes(DEGs)were identified by comparing the post-treatment with the pre-treatment transcriptomes of each patient(T1 versus TO and T2 versus TO).Quantitative PCR(qPCR)analyses were carried out for validation of RNA-Seq results.Bioinformatics and statistical analyses were conducted by using data mining approaches,such as Metascape and Ingenuity Pathway Analysis.Gene regulatory network was constructed based on DEG analysis,and critical pathways and functional genes were investigated.Serum of peripheral blood from 24 GPP patients and 28 healthy controls were collected and subjected to Luminex xMAP assays for quantitating the levels of 27 pro-inflammatory cytokines.Multivariate analysis,such as principal component analysis and hierarchical cluster analysis,were performed on deferentially expressed cytokines in serum from GPP patients and the healthy controls.Moreover,serum from GPP patients were collected at three time points:prior to treatment,treated for two weeks,and treated for six weeks.Luminex xMAP assays were performed on the collected serum for quantitating the levels of 27 pro-inflammatory cytokines.Statistical analyses were then performed to analyze the correlation between serum cytokine levels and disease severity.ResultsRNA-Seq analysis unveiled that the transcriptomic profile in five GPP patients’ PBMCs was profoundly changed following acitretin treatment.DEG analysis demonstrated that the expression of genes related to psoriatic inflamation,including CXCL1,CXCL8(IL-8),S100A8,S100A9,S100A12 and LCAN2,were significantly decreased in convalescent patients.Results from qPCR analyses confirmed the RNA-Seq data.Functional enrichment and annotation analysis revealed a cluster of DEGs significantly associated with the function of leukocytes,particularly neutrophils.Canonical pathway analysis revealed that multiple pathways,including IL-1,IL-6,IL-17A,and IFN were inhibited in patients in remission from GPP.Moreover,the expression of genes involved in innate immune response,such as TLR1,TLR5,TLR8,MYD88,NLRC4,NLRC5,NOD2,and IRF7,was deceased in convalescent patients.This analysis not only recapitulated known signaling pathways in GPP pathogenesis but also implicated novel factors and pathways,such as polo-like kinase(PLK)signaling in cell cycle regulation pathways.Regulator network analysis suggested that upstream molecules as potential therapeutic targets,such as IL-6,IL-1β,IFN-γ,IL-21,IL-5,IL-17A,TNF,and OSM.Quantitative analysis based on Luminex analysis revealed that serum levels of IL-1β(p<0.001),IL-1Ra(p<0.001),IL-4(p<0.001),IL-5(p<0.001),IL-6(p<0.001),IL-7(p<0.001),IL-8(p=0.006),IL-10(p<0.001),IL-12(p70)(p<0.001),IL-13(p<0.05),IL-17(p<0.001),G-CSF(p<0.001),IFN-y(p<0.001),IP-10(p=0.002),MCP-1(p=0.001).TNF-a(p<0.001),and VEGF(p<0.001)were found to be significantly elevated in GPP patients as compared to those in affected controls,indicating the immune response was strongly activated in GPP patients in acute phase.Serum levels of IL-2,IL-15,FGF-basic,GM-CSF,and PDGF-bb remained unchanged in GPP patients.Eotaxin level was decreased(p=0.03)in GPP patients as compared to that in controls.Serum levels of IL-6,G-CSF,IL-8,IFN-γ,IL1-Rα,IL-10,IL-13,and IL-1β showed significant correlations with the severity of GPP(r>0.4,p<0.001).Following treatment,serum levels of IL-6,IL-7,IL-12,IFN-γ,IL-1β,IL-4,IL-8,IL-10,IL-13,G-CSF,and IP-10 were significantly decreased(p<0.05)in patients.Of those,IL-6,IL-7,IL-8,IL-12,IL-13,IP-10 was found to be decreased to the normal level.The levels of IL-5,MCP-1,IL-9,IL-17,Eotaxin,TNF-a,and VEGF were not significantly changed in patients received treatment.IL-6 is the cytokine with the strongest correlation with diseases severity(r=0.683,p<0.0001)and the sole factor associated with fever in patients.Besides,the serum level of IL-6 decreased rapidly to a normal level after treatment.ConclusionsBy combining RNA-Seq and Luminex xMAP assays,together with bioinformatics and multivariate statistical analysis,this study profiled transcriptomic and cytokinomic changes associated with GPP and uncovered that gene expressions related to immune response had been profoundly altered.Gene interaction network analysis revealed that genes such as IL-6,IL-1β,and IFN-γ may play roles in driving the aberrant immune response.Moreover,specific pathways,such as innate immune response and cell cycle signaling pathways,are involved in GPP pathogenesis and remission.This study also suggested that IL-6 could be the most robust indicator for GPP diagnosis among all the cytokines/chemokines.As such,our results exemplified the value of systems approaches in studies of inflammatory disease and could provide insights into GPP pathogenesis and novel therapeutics.