The Mechanism Of MTOR Pathway Involved In Immune Dysfunction In Primary Immune Thrombocytopenia

1.Research backgroundPrimary immune thrombocytopenia(ITP)is an autoimmune disease characterized by excessive platelet destruction and decreased platelet production.The prevalence of ITP in adults is about 10/105,and the incidence rate is 1.6-3.9/105/year.B cells in patients with ITP can produce autoreactive antibodies against their own platelets.Platelet destruction in patients with ITP is mainly mediated by IgG antibodies.Antiplatelet antibodies can participate in platelet destruction through complement dependent cytotoxicity,and can also act on megakaryocytes and affect the maturation and differentiation of megakaryocytes and the production of platelets.Rituximab induces B cell lysis by combining with CD20 specifically expressed by B cells,but more than 40%of patients do not respond to rituximab treatment.Therefore,it is of great significance to seek new targets to regulate B cell autoimmune response for the treatment of ITP.Dendritic cells(DCs)are the main antigen-presenting cells in the body.Monocyte derived dendritic cells(moDCs)from ITP patients can phagocytize apoptotic platelets and stimulate the production of autoantigen reactive B cells,suggesting that the abnormal function of DCs may be an important factor in the occurrence of ITP,but its specific mechanism is not clear.To sum up,this study is divided into two parts.The first part aims to find the key molecular pathways regulating B-cell immune response in patients with ITP by using transcriptome sequencing technology,and explore whether specific molecular targeted drugs can improve B-cell autoimmune response in patients with ITP and its mechanism.In the second part,through the transcriptome sequencing of modes in patients with ITP,to explore the role and mechanism of dendritic cells in the abnormal immune response of ITP,in order to find a new treatment strategy for patients with ITP.2.Research objectives(1)The first part① Abnormal immune function of peripheral B cells in ITP patients was determined by detecting the differentiation status,transcription markers of differentiation,antibody secretion and antigen presentation costimulatory molecules.② Transcriptome sequencing technology was used to conduct bioinformatics analysis of peripheral B cells of ITP patients and health controls,and to find the key molecular pathways regulating the immune response of ITP patients B cells.③To explore the effect and mechanism of specific molecular targeted drugs on improving peripheral B cell immune dysfunction in ITP patients.(2)The second partTranscriptome sequencing technology was used to conduct bioinformatics analysis of moDCs in ITP patients and health controls,and to find the key molecular pathways regulating moDCs immune function in ITP patients.3.Research methods(1)The first part① Twenty-four ITP patients with positive monoclonal antibody specific platelet antigen fixation test and healthy volunteers with matched sex and age were divided into ITP group and health group.Flow cytometry,polymerase chain reaction and Enzyme-Linked immunosorbent assay(ELISA)were used to analyze B cell differentiation status,differentiation transcription markers,antibody production capacity and expression of antigen presentation costimulatory molecules in both groups.② Peripheral blood samples were collected from 4 ITP patients and 5 healthy volunteers with matching sex and age.CD19+B cells were selected for transcriptome sequencing.③ According to the results of differential gene analysis and enrichment analysis,the abnormal expression of mTOR signaling pathway in peripheral B cells of ITP patients was determined,and the expression of mTOR signaling pathway in peripheral B cells of ITP patients was verified by western blot and Protein Simple Wes technology.④ The half maximal inhibitory concentration of mTORC1 specific inhibitor rapamycin and mTORC1 and mTORC2 double inhibitor Torin1 on B cells were determined by Celltiter-Glo Luminescence.CD19+B cells were cultured with 1μg/ml CD40L,1Ong/ml rhIL-4,2.5μg/ml CpG,1 nM rapamycin or torin-1.B cell differentiation status,differentiation transcription markers,antibody secretion and antigen presentation costimulatory molecules were detected by flow cytometry,PCR and ELISA.(2)The second part① Peripheral blood samples from 3 ITP patients and 3 healthy volunteers with matched sex and age were collected and selected.CD14+monocytes were sorted and cultured in RPMI 1640 medium containing 100ng/ml granulocyte-macrophage colony stimulating factor and 50ng/ml IL-4 for 5 days.Then,1μg/ml lipopolysaccharide was added to induce the maturation of moDCs for 2 days.② Total RNA was extracted with Trizol reagent,and the integrity and purity of RNA were detected by agarose gel electrophoresis and ultramicro spectrophotometry.The samples with a concentration greater than 0.2μg/ml and OD260/280 in the range of 1.9-2.1 were selected for transcriptome sequencing.RNA library construction,quality inspection and transcriptome sequencing were completed by Illumina Hiseq platform of Beijing Nuohe Zhiyuan Technology Co.,LTD.③According to the results of differential gene analysis and enrichment analysis,the mTORCl signaling pathway with abnormal expression in moDCs of ITP patients was determined,and the expression of mTORC1 signaling pathway in moDCs of ITP patients was verified by western blot.4.Research results(1)The first part① Peripheral B cells of ITP patients have immune hyperfunction.The proportion of CD27+CD38+cells(plasmablasts)and the expression of BLIMP1 and IRF4 of ITP patients were significantly increased.Serum IgG and IgM antibodies were significantly increased in ITP patients,and the expression of B cell antigen presenting costimulatory molecules CD80 and CD86 was increased.② The genes in ITP group were enriched in "protein targeting to ER","protein localization to ER","mRNA catabolism of nuclear transcription" and "ribosomal structure" in BP terms,and also were enriched in "ribosomal","oxidative phosphorylation" and "protein processing in er "KEGG terms.GSEA analysis showed that mTOR signaling pathway gene expression was significantly up-regulated in peripheral CD19+B cells of ITP patients.The results showed that phosphorylated mTOR(p-MTOR),phosphorylated S6K(p-S6K)and phosphorylated Akt(p-Akt)were significantly up-regulated in peripheral B cells of ITP patients.③In B cells treated with 1nM rapamycin,mTORC1 signaling pathway was strongly inhibited,and p-S6K expression was significantly decreased,while p-Akt expression was not significantly changed.Rapamycin can reduce the proportion of plasmablasts,inhibit the expression of BLIMP1 and IRF4 in B cells,and significantly reduced the contents of IgG and IgM in cell culture supernatant.Rapamycin inhibited the expression of antigen presenting costimulatory molecules CD80 and CD86.④ 1nM Torin1 decreased the phosphorylation of mTORC1 and mTORC2 in B cells of ITP patients,resulting in a significant decrease in p-S6K and p-Akt.There was no significant difference in the inhibition of mTORCl by Troinl and rapamycin.Torin1 can reduce the proportion of plasma blasts,inhibit the expression of BLIMP1 and IRF4,and significantly reduce the contents of IgG and IgM in cell culture supernatants.Torin1 also reduced the expression of CD80 and CD86 on the surface of B cells.(2)The second part① GO and KEGG enrichment analysis results showed that the differential moDCs genes in ITP patients mainly concentrated in T cell differentiation,T cell costimulation,T cell activation and T cell receptor signaling pathway,and 10 genes were annotated in natural killer cell-mediated toxicity signaling pathway.②GSEA analysis showed that the expression of mTORC1 signaling pathway genes in moDCs of ITP patients was significantly upregulated.In addition,the gene sets of three related signaling pathways,including glycolysis,inflammatory factor reactivity and interferon y response,were significantly upregulated in moDCs of ITP patients.③ Western blot results showed that the expression of p-mTOR in moDCs of ITP patients was significantly increased,and the phosphorylation level of S6K,an activation marker of mTORC1 signaling pathway,was significantly increased.5.Research conclusions(1)The first part① Peripheral B cells of ITP patients have immune hyperfunction.② mTOR signaling pathway is highly activated in peripheral B cells of ITP patients,which may be an important target for regulating B cell immune function of ITP patients.③Rapamycin can inhibit the differentiation of B cells into plasmablasts by inhibiting mTORC1 activity,and inhibit the expression of B cell transcriptional differentiation markers and antibody secretion,and inhibit the expression of antigen presentation costimulatory molecules.④ Torin1 can inhibit the differentiation of B cells into plasmablasts by inhibiting the activity of mTORCl and mTORC2,and inhibit the expression of B cell transcriptional differentiation markers and antibody secretion,and inhibit the expression of antigen presentation costimulatory molecules.However,rapamycin and Torin1 had no significant difference in regulating the immune function of peripheral blood B cells in ITP patients.(2)The second partmoDCs in ITP patients may be involved in the pathogenesis of ITP through the immune function regulation of T cells and NK cells mediated by mTORC1 signaling pathway,and mTORC1 signaling pathway may be a new target for regulating moDCs dysfunction in ITP patients.