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Learn MorePurpose: To understand the mechanistic basis of the adjuvant role of extracellular ISG15 to support the CTL response and define whether ISG15 protein expression has a local effect on the vaccinated skin during a therapeutic DNA vaccination model, we performed mRNA deep sequencing analysis of the whole skin from the vaccinated area.; Methods: Two groups of mice (3 mice per group) were vaccinated using a tottoo device, either with pDNA encoding a shuffled version of the E7 protein alone (E7SH/EV) or combined with pDNA encoding a truncated version of ISG15 that expresses only its free form (E7SH/ISG15-GG). Vaccination was performed on days 0 and 3 and the whole skin from the vaccinated area was isolated on day 4. Differential expression of genes was assessed using the DESeq2 package. Genes with adjusted p values 0.05 were deemed significantly differentially expressed. To find activated pathways, overexpressed in comparison to the control (log-fold change > 0, adjusted p value 0.1) genes were used as input for enrichment analysis using the Reactome enrichment analysis tool. Additionally, we analysed activated pathways using Ingenuity Pathway Analysis IPA.; Results: Statistical analysis of normalized transcript read counts showed that 444 genes were differentially expressed in the skin as a result of ISG15 coexpression. Using IPA we identified 15 functional categories predicted to be activated (z-score 2) indicating that ISG15 stimulates cell migration, in particular of myeloid cells and endothelial cells. In addition, gene ontology analysis using Reactome indicated an increased metalloprotease activity, collagen degradation and extracellular matrix organization.; Conclusion: Our study represents the first analysis in an in vivo setting of ISG15 expression in keratinocytes, with biologic replicates, generated by RNA-seq technology. The results show that free ISG15 proves to be an alarmin that induces tissue alert, characterized by extracellular matrix remodelling, myeloid cell infiltration and inflammation. SOURCE: Victoria Iglesias Netherlands Cancer Institute
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