Purpose:To investigate the role of interleukin (IL)-17-producing CD4+ T cells in Behcet disease (BD).Methods:Blood samples were drawn from eight BD patients with active uveitis,eight BD patients with inactive uveitis ...Purpose:To investigate the role of interleukin (IL)-17-producing CD4+ T cells in Behcet disease (BD).Methods:Blood samples were drawn from eight BD patients with active uveitis,eight BD patients with inactive uveitis and eight normal controls,respectively.PBMCs were prepared from heparinized blood by Ficoll-Hypaque density-gradient centrifugation.Peripheral CD4+ T cells were purified by Human CD4 Microbeads.(MACS).The purity rate of CD4+ T cells was detected using flow cytometry.Purified CD4+ T cells were stimulated with or without anti-CD3 and anti-CD28 antibodies in the presence or absence of recombinant-IL-23 (rIL-23) or recombinant-IL-12 (rIL-12) for 72 hours.The concentrations of IL-17,IFN-γ and IL-4 in the collected supernatants from CD4+ T cells were measured using a Duoset ELISA Development kit.Results:The results showed that the levels of IL-17 and IFN-γ observed in active BD patients were significantly higher as compared with those in inactive patients and normal controls.There was no significant difference concerning IL-4 production between BD patients and normal controls.rIL-23 significantly augmented the production of IL-17 by CD4+ T cells from both BD patients and normal controls.Both rIL-23 and rIL-12 could increase IFN-γ production by CD4+ T cells from BD patients and normal controls.Moreover,the effect of rIL-12 was more robust compared with that of rIL-23.Neither rIL-23 nor rIL-12 exerted any effect on IL-4 production.Conclusion:rIL-23 can promote the production of IL-17 by CD4+ T cells in BD patients.The upregulated IL-17 levels may be related with the intraocular inflammation of Behcet patients.展开更多
Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing stud...Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing study,which enrolled 25 VKH patients and 50 controls,followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations.A total of 15 de novo mutations in VKH patients were identified,with one of the most important being the membrane palmitoylated protein 2(MPP2)p.K315N(MPP2-N315)mutation.The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions.Additionally,this mutation appears rare,being absent from the 1000 Genome Project and Genome Aggregation Database,and it is highly conserved in 10 species,including humans and mice.Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis(EAU).In vitro,we used clustered regularly interspaced short palindromic repeats(CRISPR‒Cas9)gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315.Levels of cytokines,such as IL-1β,IL-17E,and vascular endothelial growth factor A,were increased,and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells.Mechanistically,the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315,as shown by LC‒MS/MS and Co-IP,and resulted in activation of the ERK3/IL-17E pathway.Overall,our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.展开更多
Behcet’s disease is defined as a multisystemic inflammatory disease.Although the precise pathogenesis and etiology is still a mystery,accumulating evidence shows that genetic variants of immune-related genes have a p...Behcet’s disease is defined as a multisystemic inflammatory disease.Although the precise pathogenesis and etiology is still a mystery,accumulating evidence shows that genetic variants of immune-related genes have a profound influence on the development of Behcet’s disease.To explore the genetic factors for Behcet’s disease,our group investigated the association of Behcet’s disease with multiple immune response genes and has identified multiple Behcet’s disease-related immunoregulatory pathways in the Chinese Han population.A large number of gene polymorphisms were studied including STAT4,IL23R,CD40,CCR1/CCR3,STAT3,OPN,IL17,JAK2,MCP-1,CTLA4,PD-1,PD-L1,PD-L2,TGRBR3,CCR6,PTPN22,FCRL3,IRF5,SUMO4 and UBAC2.Significant associations were found between Behcet’s disease and STAT4,IL23R,CD40,CCR1/CCR3,STAT3,MCP-1,TGFBR3,FCRL3,SUMO4,UBAC2.These genetic predisposition studies support an important role for both lymphocyte differentiation as well as ubiquitination pathways.These findings are helpful in elucidating the pathogenesis of Behcet’s disease and hopefully will allow the development of novel treatment regimes.展开更多
Vogt-Koyanagi-Harada disease(VKH)is a rare autoimmune disease characterized by diffuse and bilateral uveitis,alopecia,tinnitus,hearing loss,vitiligo and headache.The transcriptional expression pattern of peripheral bl...Vogt-Koyanagi-Harada disease(VKH)is a rare autoimmune disease characterized by diffuse and bilateral uveitis,alopecia,tinnitus,hearing loss,vitiligo and headache.The transcriptional expression pattern of peripheral blood mononuclear cells(PBMC)in VKH remains largely unknown.In this study,mRNA sequencing was conducted in PBMC from VKH patients with active uveitis before treatment(n=7),the same patients after prednisone combined with cyclosporine treatment(n=7)and healthy control subjects strictly matched with gender and age(n=7).We found 118 differentially expressed genes(DEGs)between VKH patients and healthy control subjects,and 21 DEGs between VKH patients before and after treatment.TRIB1 was selected as a potential biomarker to monitor the development of VKH according to the mRNA sequencing.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed to predict the possible biological functions and signaling pathways of DEGs.Neutrophil degranulation,peptidase regulator activity,secretory granule membrane,cellular response to peptide,growth factor binding and cell projection membrane were enriched as GO annotations of DEGs.Arachidonic acid metabolism and mitogen-activated protein kinase(MAPK)signaling pathway were potential signaling pathways involved in pathogenesis and drug response of VKH.A protein–protein interaction(PPI)network was constructed by STRING,and colony stimulating factor 1 receptor(CSF1R)was identified as the hubgene of all DEGs by Cytoscape.The cell type presumed to contribute to the aberrant expression of DEGs was analyzed with the use of publicly available single-cell sequencing data of PBMC from a healthy donor and single-cell sequencing dataset of monocytes from VKH patients.Our findings may help to decipher the underlying cellular and molecular pathogenesis of VKH and may lead novel therapeutic applications.展开更多
基金supported by Natural Science Foundation (30801277)Project of State Key Laborato-ry of Ophthalmology (2010Q08)+1 种基金Guangdong Province Doctor Foundation (sybzzXm201014)Guangdong Province Natural Science Foundation(9151008901000102)
文摘Purpose:To investigate the role of interleukin (IL)-17-producing CD4+ T cells in Behcet disease (BD).Methods:Blood samples were drawn from eight BD patients with active uveitis,eight BD patients with inactive uveitis and eight normal controls,respectively.PBMCs were prepared from heparinized blood by Ficoll-Hypaque density-gradient centrifugation.Peripheral CD4+ T cells were purified by Human CD4 Microbeads.(MACS).The purity rate of CD4+ T cells was detected using flow cytometry.Purified CD4+ T cells were stimulated with or without anti-CD3 and anti-CD28 antibodies in the presence or absence of recombinant-IL-23 (rIL-23) or recombinant-IL-12 (rIL-12) for 72 hours.The concentrations of IL-17,IFN-γ and IL-4 in the collected supernatants from CD4+ T cells were measured using a Duoset ELISA Development kit.Results:The results showed that the levels of IL-17 and IFN-γ observed in active BD patients were significantly higher as compared with those in inactive patients and normal controls.There was no significant difference concerning IL-4 production between BD patients and normal controls.rIL-23 significantly augmented the production of IL-17 by CD4+ T cells from both BD patients and normal controls.Both rIL-23 and rIL-12 could increase IFN-γ production by CD4+ T cells from BD patients and normal controls.Moreover,the effect of rIL-12 was more robust compared with that of rIL-23.Neither rIL-23 nor rIL-12 exerted any effect on IL-4 production.Conclusion:rIL-23 can promote the production of IL-17 by CD4+ T cells in BD patients.The upregulated IL-17 levels may be related with the intraocular inflammation of Behcet patients.
基金We thank the families for participation in this study,and we thank Novogene Technology Co.,Ltd.,for the WES sequencing and analysis.This work was supported by the National Natural Science Foundation Project of China(82070951,82271078)the National Natural Science Foundation Key Program(81930023)+3 种基金The Innovative Research Group Project of Chongqing Education Commission(CXQT19015)the Innovation Supporting Plan of Overseas Study of Chongqing(cx2018010)the National Key Clinical Specialties Construction Program of China,the Chongqing Branch of the National Clinical Research Center for Ocular Diseases,the Chongqing Key Laboratory of Ophthalmology(CSTC,2008CA5003)the Program for Youth Innovation in Future Medicine,Chongqing Medical University(w0047).
文摘Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing study,which enrolled 25 VKH patients and 50 controls,followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations.A total of 15 de novo mutations in VKH patients were identified,with one of the most important being the membrane palmitoylated protein 2(MPP2)p.K315N(MPP2-N315)mutation.The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions.Additionally,this mutation appears rare,being absent from the 1000 Genome Project and Genome Aggregation Database,and it is highly conserved in 10 species,including humans and mice.Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis(EAU).In vitro,we used clustered regularly interspaced short palindromic repeats(CRISPR‒Cas9)gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315.Levels of cytokines,such as IL-1β,IL-17E,and vascular endothelial growth factor A,were increased,and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells.Mechanistically,the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315,as shown by LC‒MS/MS and Co-IP,and resulted in activation of the ERK3/IL-17E pathway.Overall,our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.
文摘Behcet’s disease is defined as a multisystemic inflammatory disease.Although the precise pathogenesis and etiology is still a mystery,accumulating evidence shows that genetic variants of immune-related genes have a profound influence on the development of Behcet’s disease.To explore the genetic factors for Behcet’s disease,our group investigated the association of Behcet’s disease with multiple immune response genes and has identified multiple Behcet’s disease-related immunoregulatory pathways in the Chinese Han population.A large number of gene polymorphisms were studied including STAT4,IL23R,CD40,CCR1/CCR3,STAT3,OPN,IL17,JAK2,MCP-1,CTLA4,PD-1,PD-L1,PD-L2,TGRBR3,CCR6,PTPN22,FCRL3,IRF5,SUMO4 and UBAC2.Significant associations were found between Behcet’s disease and STAT4,IL23R,CD40,CCR1/CCR3,STAT3,MCP-1,TGFBR3,FCRL3,SUMO4,UBAC2.These genetic predisposition studies support an important role for both lymphocyte differentiation as well as ubiquitination pathways.These findings are helpful in elucidating the pathogenesis of Behcet’s disease and hopefully will allow the development of novel treatment regimes.
基金supported by National Natural Science Foundation Key Program,China(No.81930023)Natural Science Foundation Major International(Regional)Joint Research Project,China(No.81720108009)+2 种基金Chongqing Outstanding Scientists Project(2019),Chongqing Key Laboratory of Ophthalmology,China(No.CSTC,2008CA5003)Chongqing Science&Technology Platform and Base Construction Program,China(No.cstc2014ptsy10002)the Chongqing Chief Medical Scientist Project,China(2018).
文摘Vogt-Koyanagi-Harada disease(VKH)is a rare autoimmune disease characterized by diffuse and bilateral uveitis,alopecia,tinnitus,hearing loss,vitiligo and headache.The transcriptional expression pattern of peripheral blood mononuclear cells(PBMC)in VKH remains largely unknown.In this study,mRNA sequencing was conducted in PBMC from VKH patients with active uveitis before treatment(n=7),the same patients after prednisone combined with cyclosporine treatment(n=7)and healthy control subjects strictly matched with gender and age(n=7).We found 118 differentially expressed genes(DEGs)between VKH patients and healthy control subjects,and 21 DEGs between VKH patients before and after treatment.TRIB1 was selected as a potential biomarker to monitor the development of VKH according to the mRNA sequencing.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed to predict the possible biological functions and signaling pathways of DEGs.Neutrophil degranulation,peptidase regulator activity,secretory granule membrane,cellular response to peptide,growth factor binding and cell projection membrane were enriched as GO annotations of DEGs.Arachidonic acid metabolism and mitogen-activated protein kinase(MAPK)signaling pathway were potential signaling pathways involved in pathogenesis and drug response of VKH.A protein–protein interaction(PPI)network was constructed by STRING,and colony stimulating factor 1 receptor(CSF1R)was identified as the hubgene of all DEGs by Cytoscape.The cell type presumed to contribute to the aberrant expression of DEGs was analyzed with the use of publicly available single-cell sequencing data of PBMC from a healthy donor and single-cell sequencing dataset of monocytes from VKH patients.Our findings may help to decipher the underlying cellular and molecular pathogenesis of VKH and may lead novel therapeutic applications.
