Selective Whole-Blood Expression of Interferon-Stimulated Genes and TLR3/TLR7 in Type 1 Diabetes with Minimal Enterovirus-Targeted Amplification in Whole Blood

Abstract

Background: Enteroviruses (EVs) have been proposed as environmental triggers in type 1 diabetes (T1D), potentially through activation of innate immune pathways and type I interferon (IFN) responses. However, the relationship between detectable viral infection and IFN-related transcriptional responses remains unclear. Methods: In this cross-sectional, exploratory study, peripheral blood samples from individuals with new-onset T1D, established T1D, and healthy controls were analyzed by an enterovirus 5′NCR-targeted RT-PCR assay and for the expression of innate immune receptors (TLR3, TLR7, TLR8), interferon-stimulated genes (IFI27, IFIT1, ISG15, IFI44L, RSAD2, SIGLEC1), and FOXP3, assessed as an exploratory marker related to regulatory T-cell biology potentially influenced by type I interferon signaling. Gene expression was assessed by quantitative RT-PCR, and comparisons were performed between groups. Results: Enterovirus-targeted amplification was observed in only one T1D sample under the applied assay conditions. In the setting of limited enterovirus-targeted amplification under the applied assay conditions, TLR3 was significantly increased in established T1D, while TLR7 was increased in both new-onset and established T1D, with the highest levels in established disease; these differences remained significant after correction for multiple comparisons across the full gene panel. TLR8 was unchanged. Several of the selected interferon-stimulated genes showed group-specific expression patterns: IFI27 and RSAD2 were significantly increased in new-onset T1D, whereas IFIT1 and ISG15 were more evident in established T1D. SIGLEC1 and IFI44L showed no significant differences. FOXP3 transcript abundance was also increased in both diabetic groups, a finding that correlated with lymphocyte proportion and is interpreted with caution. Conclusions: These findings indicate group-specific differences in whole-blood transcript expression of selected RNA-sensing and IFN-related genes in T1D under conditions in which enterovirus-targeted amplification was observed in only one sample. Given the cross-sectional whole-blood design and the analytical limitations of the EV assay, these results are hypothesis-generating and do not establish the presence or absence of systemic enterovirus infection or functional pathway activation.