oa Human islets-clusters express coxsackie-adenovirus and decay accelerating receptors and reveals a distinctive microRNA disease-associated signature in T1D cellular models

Human Enterovirus (HEV) infections, speci?cally Coxsackievirus B(CVB), demonstrate ß-cell tropism and are associated with type 1 diabetes (T1D). Previous studies have shown that Coxsackie-Adenovirus receptors (CAR) and Decay Accelerating Factor (DAF) are both required to initiate CVB infection in human and rodent models, but this has not been examined in Human Islets-Clusters (HICS), which are derived from human islet progenitor cells. MicroRNAs function as translational repressors and are important regulators of key biological processes, although their role in virus induced ß-cell death has not been examined. We hypothesise that (i) CBV infection of HICS is associated with up-regulation of CAR and DAF receptors and down-regulation of insulin and pdx-1 genes; ß-cell death and (ii) CVB infection of HICS alters microRNA abundance, thereby regulating gene expression. Purified cultured (HICS) and human islets were infected with CVB3, CVB4 and CVB5. Human microRNAs (n=756) were quanti?ed. miRBase and microRWalk algorithms were used to predict microRNA gene targets. Spearman's correlation coefficient was used to calculate the pair wise correlation between each pair of microRNAs. Hierarchical clustering was used to determine groups of microRNAs with similar expression patterns following EV infection. R software was used for analyses and for creating heat maps. We identified 21 microRNAs associated with T1D candidate genes that were increased > 10 fold (relative to uninfected controls, p<0.05) following CVB infection of human islets. In the HICS, 23 microRNAs were differentially-expressed after CVB5 infection. Many of the microRNAs target genes that control cytokine production and signalling (eg IL-2, IL-2RA, IL-10, PTPN22), T cell receptor signalling (PRKCQ, RASGRP1), immune response to viral infection (TNFAIP3) and apoptosis (TYK2). Heat maps demonstrated two clusters: 8 microRNAs increased by CVB3, 4, 5) and 13 microRNAs (CVB3 only). Analysis of interactions between microRNAs with >10 fold higher expression post CBV infection and a human T1D protein network showed microRNAs mainly target positive regulatory motifs in highly connected scaffolds. The changes in the expression levels of (CAR and DAF), insulin and Pdx-1 genes in the infected cells were analysed with TaqMan real-time PCR. HEV specific capsid protein (VP1) was also measured . Infected and non-infected HICS were stained with VP1, CAR and DAF receptors, Pdx-1 and insulin gene specific antibodies. CVB3, 4, and 5 infected and replicated in the HICS, which expressed both CAR and DAF receptors, and remained intact with no apparent Cytopathic effect for up to 20 days post infection. Infected HICS did not show any changes in the expression levels of insulin and Pdx-1 genes at day 3 post infection. These results indicate that CBV infection of human islets and HICS leads to dysregulation of multiple microRNAs. This appears to disrupt the protection of cellular integrity, with alterations in the immune response, ultimately leading to ß-cell death. Moreover, HICS are natural targets and reservoirs for persistent CVB infection. insulin expression doesn't appear to be affected at the initial stage of infection. Thus HICS may be a useful cellular model for examining HEV infection of ß-cells and may further our understanding the virus induced diabetes.