ena-STUDY-GERMANS TRIAS I PUJOL UNIVERSITY HOSPITAL-21-08-2017-18:31:39:839-281
RNA-seq of human dendritic cells from patients with type 1 diabetes after phagocytosis of autoantigen-loaded phosphatidylserine-liposomes
Phagocytosis of apoptotic cells by dendritic cells (DCs) induces immunological tolerance. We have proved that liposomes that mimic apoptotic beta-cells can halt the autoimmune attack in a mouse model of type 1 diabetes (T1D) through the generation of tolerogenic DCs. Aiming for the clinical potential of this strategy, the goal of this study was to characterize gene expression changes in DCs from patients with T1D, 4 hours after the phagocytosis of autoantigen-loaded phosphatidylserine-liposomes. Monocyte-derived DCs were obtained from 8 patients with T1D, and DCs were co-cultured with phosphatidylserine-liposomes encapsulating insulin peptides (PSAB-DCs). Unloaded DCs (iDCs) were used as control in 8 paired experiments. RNA was extracted and used to prepare RNA libraries, which were sequenced in a NextSeq 500 genetic analyzer, obtaining 20 million reads for each sample. Forward and Reverse Fastq files were merged for each sample, and read alignment and mapping steps to gene regions were performed using CLCBio software. Data were normalized by applying the standard “Reads Per Kilobase of transcript per Million reads mapped” (RPKM) method and transformed to log2. Genes with p value <0.0013, adjusted p-values of =0.125 and LogFC >0.05 were considered upregulated, whereas those with LogFC <?0.05 were considered downregulated. Gene expression profile of DCs from patients with T1D upon capture of liposomes showed differentially expressed genes mainly involved in vesicle transport, immune response and cytokine signaling, which promote tolerogenic features. Thus, molecular changes in DCs caused by the uptake of phosphatidylserine-rich liposomes loaded with autoantigenic peptides are key factors to induce antigen-specific peripheral tolerance.
Phagocytosis of apoptotic cells by dendritic cells (DCs) induces immunological tolerance. We have proved that liposomes that mimic apoptotic beta-cells can halt the autoimmune attack in a mouse model of type 1 diabetes (T1D) through the generation of tolerogenic DCs. Aiming for the clinical potential of this strategy, the goal of this study was to characterize gene expression changes in DCs from patients with T1D, 4 hours after the phagocytosis of autoantigen-loaded phosphatidylserine-liposomes. Monocyte-derived DCs were obtained from 8 patients with T1D, and DCs were co-cultured with phosphatidylserine-liposomes encapsulating insulin peptides (PSAB-DCs). Unloaded DCs (iDCs) were used as control in 8 paired experiments. RNA was extracted and used to prepare RNA libraries, which were sequenced in a NextSeq 500 genetic analyzer, obtaining 20 million reads for each sample. Forward and Reverse Fastq files were merged for each sample, and read alignment and mapping steps to gene regions were performed using CLCBio software. Data were normalized by applying the standard “Reads Per Kilobase of transcript per Million reads mapped” (RPKM) method and transformed to log2. Genes with p value <0.0013, adjusted p-values of =0.125 and LogFC >0.05 were considered upregulated, whereas those with LogFC <?0.05 were considered downregulated. Gene expression profile of DCs from patients with T1D upon capture of liposomes showed differentially expressed genes mainly involved in vesicle transport, immune response and cytokine signaling, which promote tolerogenic features. Thus, molecular changes in DCs caused by the uptake of phosphatidylserine-rich liposomes loaded with autoantigenic peptides are key factors to induce antigen-specific peripheral tolerance.