Detection of Antiviral Tissue Responses and Increased Cell Stress in the Pancreatic Islets of Newly Diagnosed Type 1 Diabetes Patients: Results From the DiViD Study

Abstract

Aims/hypothesis: The Diabetes Virus Detection (DiViD) study has suggested the presence of low-grade enteroviral infection in pancreatic tissue collected from six of six live adult patients newly diagnosed with type 1 diabetes. The present study aimed to compare the gene and protein expression of selected virally induced pathogen recognition receptors and interferon stimulated genes in islets from these newly diagnosed type 1 diabetes (DiViD) subjects vs age-matched non-diabetic (ND) controls.

Methods: RNA was extracted from laser-captured islets and Affymetrix Human Gene 2.0 ST arrays used to obtain gene expression profiles. Lists of differentially expressed genes were subjected to a data-mining pipeline searching for enrichment of canonical pathways, KEGG pathways, Gene Ontologies, transcription factor binding sites and other upstream regulators. In addition, the presence and localisation of specific viral response proteins (PKR, MxA and MDA5) were examined by combined immunofluorescent labelling in sections of pancreatic tissue.

Results: The data analysis and data mining process revealed a significant enrichment of gene ontologies covering viral reproduction and infectious cycles; peptide translation, elongation and initiation, as well as oxidoreductase activity. Enrichment was identified in the KEGG pathways for oxidative phosphorylation; ribosomal and metabolic activity; antigen processing and presentation and in canonical pathways for mitochondrial dysfunction, oxidative phosphorylation and EIF2 signaling. Protein Kinase R (PKR) expression did not differ between newly diagnosed type 1 diabetes and ND islets at the level of total RNA, but a small subset of β-cells displayed markedly increased PKR protein levels. These PKR+ β-cells correspond to those previously shown to contain the viral protein, VP1. RNA encoding MDA5 was increased significantly in newly diagnosed type 1 diabetes islets, and immunostaining of MDA5 protein was seen in α- and certain β-cells in both newly diagnosed type 1 diabetes and ND islets, but the expression was increased in β-cells in type 1 diabetes. In addition, an uncharacterised subset of synaptophysin positive, but islet hormone negative, cells expressed intense MDA5 staining and these were more prevalent in DiViD cases. MxA RNA was upregulated in newly diagnosed type 1 diabetes vs ND islets and MxA protein was detected exclusively in newly diagnosed type 1 diabetes β-cells.

Conclusion/interpretation: The gene expression signatures reveal that pathways associated with cellular stress and increased immunological activity are enhanced in islets from newly diagnosed type 1 diabetes patients compared to controls. The increases in viral response proteins seen in β-cells in newly diagnosed type 1 diabetes provide clear evidence for the activation of IFN signalling pathways. As such, these data strengthen the hypothesis that an enteroviral infection of islet β-cells contributes to the pathogenesis of type 1 diabetes.

Keywords: MDA5; MxA; PKR; Type 1 diabetes; biopsy; enterovirus; gene expression; pancreas.

Figure 1

Micrographs illustrating the immunofluorescent staining of representative islets from an individual without diabetes (ND; column 1) and two islets from a DiViD patient with diabetes, either with or without residual beta cells (columns 2 and 3 respectively). Green, MxA; Red, Glucagon; Cyan, Insulin; Blue, DAPI. Scale bars, 25um.

Figure 2

Micrographs showing immunofluorescence images of representative islets from an individual without diabetes (ND; column 1) and from two different DiViD patients with diabetes (columns 2 & 3). PKR positive beta cells are indicated (yellow lines). Green, PKR; Red, Glucagon; Cyan, Insulin; Blue, DAPI. Scale bars, 25um.

Figure 3

Micrographs showing immunofluorescent images of a representative islet from an individual without diabetes (ND; column 1) and from two DiViD patients with diabetes (columns’ 2-3). MDA5+ alpha cells are indicated with arrows and MDA5+ beta cells with dotted lines (columns 2 & 3). Green, MDA5; Red, glucagon; Cyan, Insulin; Blue, DAPI. Scale bars, 25um.

Figure 4

Examples of MDA5+ cells which are synaptophysin + but hormone negative. Green, MDA5; Red, synaptophysin; Cyan, hormone cocktail; insulin, glucagon and somatostatin. Scale bars, 25um.