The Role Of IFN-α/β-JAK-STAT-cellular Senescence Pathway In The Pathogenesis Of Discoid Lupus Erythematosus

Background:Lupus erythematosus(LE)is a highly heterogeneous autoimmune disease.There are many differences in the organ injury site,disease process,severity,treatment response and prognosis.Skin is one of the most frequently affected organs of LE.It is still unclear what are the specific molecular pathways in the process from the initiation of autoimmune reaction to the formation of skin damage.The correlation between anti-ds DNA antibody and acute cutaneous LE(ACLE),anti-Ro antibody and subacute cutaneous LE(SCLE)has been suggested in previous studies.As for discoid lupus erythematosus(DLE),the relationship between specific autoantibodies and its characteristic lesions is unclear.The imbalance of type I interferon axis has been considered to play a key role in the pathogenesis of LE.Increasingly evidence show that tissue derived type I interferon(IFN-Ⅰ)may play a greater role in the formation of local damage of disease target organs than circulating IFN-Ⅰ.Our previous work found that there was a local immune disorder in the skin lesions of localized DLE(IDLE)patients,differing from the ACLE lesion.To date,there is no research to explore the role of type I interferon signal in the localized skin damage of IDLE.Objective:To study the immunological characteristics of IDLE skin lesions and the role of type I interferon in the formation of IDLE skin lesion.Methods:(1)The immunocyte subclass staining system,the B cell subgroup staining system and the immunoglobulin Ig G subclass staining system were constructed by the tissue multiple fluorescence immunohistochemistry technique.We provide detailed description for the immunological characteristics of IDLE lesions,which were different from ACLE.The expression and distribution of type I interferon signal in the IDLE lesions were detected by immunofluorescence staining;(2)The differentially expressed genes of DLE patients and healthy control(HC)were shown by the data mining of RNA-seq dataset(skin samples from DLE and HC)in GEO database.Protein-Protein interaction network(PPI),KEGG PATHWAY and Gene Set Enrichment Analysis(GSEA)were performed to explore the molecular pathway related to type I interferon;(3)Through quantitative real time PCR(q PCR),we researched the effect of IFN-α/βcombined stimulation on human keratinocytes.The m RNA expression of JAK-STAT pathway related genes,cell senescence and SASP related genes and chemokine related genes were evaluated.By the cell cycle detection and galactosidase staining,the cell senescence of Ha Ca T cells and human primary keratinocytes was researched.(4)DLE model mice was established by the intradermal injection of pristane combined with UVB irradiation.The composition and distribution of immune cells in the skin lesions of DLE model mice were evaluated by multiple fluorescence immunohistochemistry technique and multispectral flow cytometry.During the construction of DLE model mice,anti IFNAR antibody was injected intraperitoneally to evaluate the effect of type I interferon signal blocking on the formation of skin damage;By the q PCR and multiple fluorescence immunohistochemistry,we studied the effects on the type I interferon signal pathway after the blockage of IFNAR in vivo.Results:(1)The main immunological characteristics of IDLE lesions were as follows:significant T and B cell aggregation around hair follicle appendages;the occurrence of ectopic germinal center structure(well-circumscribed aggregates of T cells with B cells);B cells increased significantly at different development phases(na(?)ve B cells,GC B cells,memory B cells and plasma cells)(IDLE,n=8;ACLE,n=10,HC,n=10;IDLE vs.ACLE,P<0.0001;IDLE vs.HC,P<0.0001;ACLE vs.HC,P>0.05).In addition,the positive signals of total Ig G,C4b and membrane attack complex(MAC)were uniformly concentrated in the hair follicle appendages of IDLE lesions.The staining of Ig G subclasses showed that Ig G1,Ig G2 and Ig G3 aggregated around IDLE hair follicles,especially for Ig G1 and Ig G2.The strong positive signals of IFN-α,IFN-βand IFNAR1 were observed in IDLE skin lesions,and mainly concentrated in the epidermis and hair follicle appendages;(3)The PPI analysis showed that there was a close interaction between interferon stimulated genes(OASL,ISG20,ISG15,IFI6,IFIT3,etc.)and signal transducer and activator of transcription(STAT1 and STAT2),between STAT1 and chemokines(CXCL9,CXCL10,CXCL11,CXCL13,CCL4,CCL5,CCL8).In the comparison of DLE and ACLE lesions,JAK-STAT signal pathway and cellular senescence signal pathway were significantly up-regulated in DLE lesions.The result of GSEA showed the correlation between JAK-STAT and cellular senescence signal pathway;(3)IFN-α/βcombined stimulation on human Ha Cat cell line and human primary keratinocytes,activated the JAK-STAT signal pathway(JAK1,TYK2,STAT1,STAT2,IRF9),up-regulated the gene of P21,CXCL10 and CXCL13(IFN-α+IFN-βvs.Ctrl:(Ha Cat cell),n=3,JAK1,TYK2,STAT1,STAT2,CXCL10,CXCL13,P<0.01;P21,P<0.05;IRF9,P<0.0001.IFN-α+IFN-βvs.Ctrl:(human primary keratinocytes),n=3,JAK1,STAT1,P<0.05;CXCL10,P<0.01;STAT2,P<0.001;P21,P=0.0868),and acquired the characteristics senescence cell.(4)There were significant increased CD4~+T cells,total B cells,Tfh-like cells,na(?)ve B cells,memory B cells,GC B cells,plasma cells and age-associated B cell(ABC)in the lesions of DLE model mice.Besides,the results of multiple fluorescent immunohistochemistry showed that increased T and B cells were mainly surrounded hair follicles.After the blockage of IFNAR in vivo,the skin damage area of DLE model mice significantly decreased,and we also observed the reduced inflammation around hair follicles;After the blockage of IFNAR in vivo,we found the down-regulation of JAK-STAT signal pathway related genes(Jak1,Tyk2,Stat1,Stat2,Irf9),cellular senescence related genes(p16,p21,p27),chemokine CXC subfamily related genes(Cxcl2,Cxcl3,Cxcl9,Cxcl10,Cxcl11,Cxcl12,Cxcl13),chemokine CC subfamily related genes(Ccl4,Ccl5,Ccl8,Ccl11,Ccl19),senescence related secretory phenotype(Il1a,Il1B,Il6,Mcp1,Tnfa)(n=5,Pristane+Ab vs.Pristane+isotype:Oasl1,Ifit2,Isg15,Jak1,Stat1,Stat2,Irf9,p16,Il1b,Cxcl10,Cxcl13,P<0.05;Mx1,Oas2,Tyk2,p21,Mcp1,Ccl4,Ccl5,Ccl8,P<0.01;Tnfa,Cxcl9,Cxcl11,P<0.001).Conclusion:IFN-Ⅰmay promote the cellular senescence of keratinocytes by activating JAK-STAT signal pathway,contributing to the formation of skin damages of IDLE.