Enterovirus-associated changes in blood transcriptomic profiles of children with genetic susceptibility to type 1 diabetes

Abstract

Aims/hypothesis: Enterovirus infections have been associated with the development of type 1 diabetes in multiple studies, but little is known about enterovirus-induced responses in children at risk for developing type 1 diabetes. Our aim was to use genome-wide transcriptomics data to characterise enterovirus-associated changes in whole-blood samples from children with genetic susceptibility to type 1 diabetes.

Methods: Longitudinal whole-blood samples (356 samples in total) collected from 28 pairs of children at increased risk for developing type 1 diabetes were screened for the presence of enterovirus RNA. Seven of these samples were detected as enterovirus-positive, each of them collected from a different child, and transcriptomics data from these children were analysed to understand the individual-level responses associated with enterovirus infections. Transcript clusters with peaking or dropping expression at the time of enterovirus positivity were selected as the enterovirus-associated signals.

Results: Strong signs of activation of an interferon response were detected in four children at enterovirus positivity, while transcriptomic changes in the other three children indicated activation of adaptive immune responses. Additionally, a large proportion of the enterovirus-associated changes were specific to individuals. An enterovirus-induced signature was built using 339 genes peaking at enterovirus positivity in four of the children, and 77 of these genes were also upregulated in human peripheral blood mononuclear cells infected in vitro with different enteroviruses. These genes separated the four enterovirus-positive samples clearly from the remaining 352 blood samples analysed.

Conclusions/interpretation: We have, for the first time, identified enterovirus-associated transcriptomic profiles in whole-blood samples from children with genetic susceptibility to type 1 diabetes. Our results provide a starting point for understanding the individual responses to enterovirus infections in blood and their potential connection to the development of type 1 diabetes.

Data availability: The datasets analysed during the current study are included in this published article and its supplementary information files ( www.btk.fi/research/computational-biomedicine/1234-2 ) or are available from the Gene Expression Omnibus (GEO) repository (accession GSE30211).

Keywords: Clinical immunology; Enterovirus; Human; Microarray; Prediction and prevention of type 1 diabetes.

Fig. 1

(a–g) Average expression profiles of clusters peaking or dropping at enterovirus positivity for the seven enterovirus-positive children: Strong 1 (a), Strong 2 (b), Strong 3 (c), Strong 4 (d), Strong 5 (e), Weak 1 (f) and Weak 2 (g). Red, peaking clusters; blue, dropping clusters. EV+, enterovirus-positive. (h, i) Overlapping probe sets between the peaking and dropping clusters. The black areas indicate the proportion of overlapping probe sets relative to the child/cluster noted at the top of the column. The boxes highlighted in the outlined frame show the peaking (h) and dropping (i) clusters of the four strongly enterovirus-positive children with the most similar enterovirus-associated changes. The total numbers of probe sets in each cluster are presented in the diagonal. The five children with strongly enterovirus-positive blood samples are denoted as Strong 1–Strong 5. The two children with weakly enterovirus-positive blood samples are denoted as Weak 1 and Weak 2

Fig. 2

(a) Genes in peaking clusters mapping to the interferon signalling pathway based on the IPA tool. Red, genes present in at least four peaking clusters of the strongly enterovirus-positive children; pink, genes present in at least one peaking cluster. (b–h) Child-specific expression profiles of two genes of the interferon signalling pathway, MX1 (grey) and STAT2 (black). IFNγ is also known as IFNG; IFNα/β is also known as IFNA1/B1; TC-PTP is also known as PTPN2; NF-κB p65 is also known as RELA; BCL-2 is also known as BCL2; BAK is also known as BAK1; DRIP150 is also known as MED14; G1P2 is also known as ISG15; G1P3 is also known as IFI6. EV+, enterovirus-positive; GAS, IFNG-activated sequence; ISRE, interferon-stimulated regulatory element. The five children with strongly enterovirus-positive blood samples are denoted as Strong 1–Strong 5 (b–f). The two children with weakly enterovirus-positive blood samples are denoted as Weak 1 and Weak 2 (g, h)

Fig. 3

Expression of the 77 genes present in our enterovirus-induced signature and upregulated in all three in vitro enterovirus infections in microarray data published by: (a) Kallionpää et al [15]; and (b) Ferreira et al [27]. Sums of child-specific z scores over the 77 genes were calculated for each of the 356 whole blood samples by Kallionpää et al [15] (GEO: GSE30211) and the 454 PBMC samples by Ferreira et al [27] (Array Express: E-MTAB-1724), as described in Methods, using the published pre-processed datasets and sample information based on personal communications with Ferreira et al. All probes (a) or the highest-intensity exons mapping to genes (b) overlapping with the 77 genes were summed. (a) Black, strongly enterovirus-positive blood samples; white, weakly enterovirus-positive blood samples; grey, enterovirus-negative blood samples. (a, b) PreSero, samples collected from before seroconversion from children with autoantibody positivity or type 1 diabetes (in a, n = 22; in b, n = 65); PostSero, samples collected after seroconversion from children with autoantibody positivity or type 1 diabetes (in a, n = 169; b n = 84), Aab⁻, samples collected from autoantibody-negative children (in a, n = 165; in b, n = 305). (c) Venn diagram showing the overlaps between the 77 genes present in the enterovirus-induced signature and upregulated in all three in vitro enterovirus infections; genes upregulated before or after seroconversion in autoantibody-positive children based on the results by Kallionpää et al [15] and the 225 interferon-inducible genes detected by Ferreira et al [27]