Detection of enterovirus RNA in peripheral blood mononuclear cells correlates with the presence of the predisposing allele of the type 1 diabetes risk gene IFIH1 and with disease stage

Aims/hypothesis Enteroviral infection has been implicated consistently as a key environmental factor correlating with the appearance of autoimmunity and/or the presence of overt type 1 diabetes, in which pancreatic insulin-producing beta cells are destroyed by an autoimmune response. Genetic predisposition through variation in the type 1 diabetes risk gene IFIH1 (interferon induced with helicase C domain 1), which encodes the viral pattern-recognition receptor melanoma differentiation-associated protein 5 (MDA5), supports a potential link between enterovirus infection and type 1 diabetes. Methods We used molecular techniques to detect enterovirus RNA in peripheral blood samples (in separated cellular compartments or plasma) from two cohorts comprising 79 children or 72 adults that include individuals with and without type 1 diabetes who had multiple autoantibodies. We also used immunohistochemistry to detect the enteroviral protein VP1 in the pancreatic islets of post-mortem donors (n=43) with type 1 diabetes. Results We observed enhanced detection sensitivity when sampling the cellular compartment compared with the non-cellular compartment of peripheral blood (OR 21.69; 95% CI 3.64, 229.20; p Conclusions/interpretation Our data indicate that, in peripheral blood, antigen-presenting cells are the predominant source of enterovirus infection, and that infection is correlated with disease stage and genetic predisposition, thereby supporting a role for enterovirus infection prior to disease onset.Peer reviewe

Detection of enterovirus RNA in peripheral blood mononuclear cells correlates with the presence of the predisposing allele of the type 1 diabetes risk gene IFIH1 and with disease stage

Aims/hypothesis Enteroviral infection has been implicated consistently as a key environmental factor correlating with the appearance of autoimmunity and/or the presence of overt type 1 diabetes, in which pancreatic insulin-producing beta cells are destroyed by an autoimmune response. Genetic predisposition through variation in the type 1 diabetes risk gene IFIH1 (interferon induced with helicase C domain 1), which encodes the viral pattern-recognition receptor melanoma differentiation-associated protein 5 (MDA5), supports a potential link between enterovirus infection and type 1 diabetes.Methods We used molecular techniques to detect enterovirus RNA in peripheral blood samples (in separated cellular compartments or plasma) from two cohorts comprising 79 children or 72 adults that include individuals with and without type 1 diabetes who had multiple autoantibodies. We also used immunohistochemistry to detect the enteroviral protein VP1 in the pancreatic islets of post-mortem donors (n=43) with type 1 diabetes.Results We observed enhanced detection sensitivity when sampling the cellular compartment compared with the non-cellular compartment of peripheral blood (OR 21.69; 95% CI 3.64, 229.20; p</p

DNA methylation differences within INS, PTPN22 and IL2RA promoters in lymphocyte subsets in children with type 1 diabetes and controls

Abstract We elucidated the effect of four known T1D-susceptibility associated single nucleotide polymorphism (SNP) markers in three genes (rs12722495 and rs2104286 in IL2RA, rs689 in INS and rs2476601 in PTPN22) on CpG site methylation of their proximal promoters in different lymphocyte subsets using pyrosequencing. The study cohort comprised 25 children with newly diagnosed T1D and 25 matched healthy controls. The rs689 SNP was associated with methylation at four CpG sites in INS promoter: −234, −206, −102 and −69. At all four CpG sites, the susceptibility genotype AA was associated with a higher methylation level compared to the other genotypes. We also found an association between rs12722495 and methylation at CpG sites −373 and −356 in IL2RA promoter in B cells, where the risk genotype AA was associated with lower methylation level compared to the AG genotype. The other SNPs analyzed did not demonstrate significant associations with CpG site methylation in the examined genes. Additionally, we compared the methylation between children with T1D and controls, and found statistically significant methylation differences at CpG −135 in INS in CD8+ T cells (p = 0.034), where T1D patients had a slightly higher methylation compared to controls (87.3 ± 7.2 vs. 78.8 ± 8.9). At the other CpG sites analyzed, the methylation was similar. Our results not only confirm the association between INS methylation and rs689 discovered in earlier studies but also report this association in sorted immune cells. We also report an association between rs12722495 and IL2RA promoter methylation in B cells. These results suggest that at least part of the genetic effect of rs689 and rs12722495 on T1D pathogenesis may be conveyed by DNA methylation

HLA‐DQ‐conferred risk for type 1 diabetes does not alter neutralizing antibody response to a widely used enterovirus vaccine, the poliovirus vaccine

Abstract This study investigated whether children with HLA-DQ-conferred risk for type 1 diabetes (T1D) have an altered immune response to the widely-used enterovirus vaccine, namely poliovirus vaccine, and whether initiation of autoimmunity to pancreatic islets modulates this response. Neutralizing antibodies induced by the inactivated poliovirus vaccine against poliovirus type 1 (Salk) were analysed as a marker of protective immunity at the age of 18 months in a prospective birth cohort. No differences were observed in antibody titers between children with and without genetic risk for T1D (odds ratio [OR] = 0.90 [0.83, 1.06], p = 0.30). In the presence of the genetic risk, no difference was observed between children with and without islet autoimmunity (OR = 1.00 [0.78, 1.28], p = 1.00). This did not change when only children with the autoimmunity before 18 months of age were included in the analyses (OR = 1.00 [0.85, 1.18], p = 1.00). No effect was observed when groups were stratified based on autoantigen specificity of the first-appearing autoantibody (IAA or GADA). The children in each comparison group were matched for sex, calendar year and month of birth, and municipality. Accordingly, we found no indication that children who are at risk to develop islet autoimmunity would have a compromised humoral immune response which could have increased their susceptibility for enterovirus infections. In addition, the proper immune response supports the idea of testing novel enterovirus vaccines for the prevention of T1D among these individuals

Heterogeneity in the presentation of clinical type 1 diabetes defined by the level of risk conferred by human leukocyte antigen class II genotypes

Abstract Objectives: The association between human leukocyte antigen (HLA) class II genotypes and susceptibility to type 1 diabetes (T1D) is well established. This study aimed at examining whether there are differences in the presentation of T1D depending on the HLA genotype. Research Design and Methods: We divided the study participants (N = 5798) in the Finnish Pediatric Diabetes Register into two groups based on the T1D risk conferred by their HLA genotype (high and moderate-risk genotypes, Group 1 vs. other genotypes, Group 2). We then examined differences in clinical, metabolic, and immunological characteristics. Children included in the study were 0–14-year-old and diagnosed between January 2003 and December 2019. Results: Participants in Group 1 were younger at the time of diagnosis (P &lt; 0.001) and had more frequently family members affected by T1D (P &lt; 0.001). Diabetic ketoacidosis (DKA) was more frequent among participants in Group 2 (P = 0.014) who also had a longer duration of symptoms before diagnosis (P &lt; 0.001) and higher hemoglobin A1c (P = 0.001) at diagnosis. The HLA genotype was not, however, directly related to the DKA frequency. The frequency of islet cell antibodies (P &lt; 0.003), insulin autoantibodies (P &lt; 0.001), and islet antigen 2 autoantibodies (P &lt; 0.001) was higher in Group 1 whereas glutamic acid decarboxylase autoantibodies were more frequent (P &lt; 0.001) in Group 2. Group 1 had more participants with multiple autoantibodies (P = 0.027) whereas antibody negativity was more frequent in Group 2 (P = 0.003). Conclusions: These findings indicate disease heterogeneity in relation to both clinical disease presentation and humoral autoimmunity, in particular. This heterogeneity is, at least partly, defined by HLA Class II genotypes

Viral infection-related gene upregulation in monocytes in children with signs of β-cell autoimmunity

Abstract Objective: The pathogenesis of type 1 diabetes (T1D) is associated with genetic predisposition and immunological changes during presymptomatic disease. Differencesin immune cell subset numbers and phenotypes between T1D patients and healthy controls have been described; however, the role and function of these changes in the pathogenesis is still unclear. Here we aimed to analyze the transcriptomic landscapes of peripheral blood mononuclear cells (PBMCs) during presymptomatic disease. Methods: Transcriptomic differences in PBMCs were compared between cases positive for islet autoantibodies and autoantibody negative controls (9 case–control pairs)and further in monocytes and lymphocytes separately in autoantibody positive subjects and control subjects (25 case–control pairs). Results: No significant differential expression was found in either data set. However, when gene set enrichment analysis was performed, the gene sets “defence response to virus” (FDR &lt;0.001, ranking 2), “response to virus” (FDR &lt;0.001, ranking 3) and “response to type I interferon” (FDR=0.002, ranking 12) were enriched in the upregulated genes among PBMCs in cases. Upon further analysis, this was also seen in monocytes in cases (FDR=0.01, ranking 2; FDR=0.04, ranking 3 and FDR=0.02, ranking 1, respectively) but not in lymphocytes. Conclusion: Gene set enrichment analysis of children with T1D-associated autoimmunity revealed changes in pathways relevant for virus infection in PBMCs, particularly in monocytes. Virus infections have been repeatedly implicated in the pathogenesis of T1D. These results support the viral hypothesis by suggesting altered immune activation of viral immune pathways in monocytes during diabetes

Type 1 diabetes linked PTPN22 gene polymorphism is associated with the frequency of circulating regulatory T cells

Abstract Dysfunction of FOXP3‐positive regulatory T cells (Tregs) likely plays a major role in the pathogenesis of multiple autoimmune diseases including type 1 diabetes (T1D). Whether genetic polymorphisms associated with the risk of autoimmune diseases affect Treg frequency or function is currently unclear. Here, we analysed the effect of T1D‐associated major HLA class II haplotypes and seven single nucleotide polymorphisms in six non‐HLA genes [INS (rs689), PTPN22 (rs2476601), IL2RA (rs12722495 and rs2104286), PTPN2 (rs45450798), CTLA4 (rs3087243), and ERBB3 (rs2292239)] on peripheral blood Treg frequencies. These were determined by flow cytometry in 65 subjects who had progressed to T1D, 86 islet autoantibody‐positive at‐risk subjects, and 215 islet autoantibody‐negative healthy controls. The PTPN22 rs2476601 risk allele A was associated with an increase in total (p = 6 × 10⁻⁶) and naïve (p = 4 × 10⁻⁵) CD4+CD25+CD127lowFOXP3+ Treg frequencies. These findings were validated in a separate cohort comprising ten trios of healthy islet autoantibody‐negative children carrying each of the three PTPN22 rs2476601 genotypes AA, AG, and GG (p = 0.005 for total and p = 0.03 for naïve Tregs, respectively). In conclusion, our analysis implicates the autoimmune PTPN22 rs2476601 risk allele A in controlling the frequency of Tregs in human peripheral blood

Circulating metabolic signatures of rapid and slow progression to type 1 diabetes in islet autoantibody-positive children

Abstract Aims/hypothesis: Appearance of multiple islet cell autoantibodies in early life is indicative of future progression to overt type 1 diabetes, however, at varying rates. Here, we aimed to study whether distinct metabolic patterns could be identified in rapid progressors (RP, disease manifestation within 18 months after the initial seroconversion to autoantibody positivity) vs. slow progressors (SP, disease manifestation at 60 months or later from the appearance of the first autoantibody). Methods: Longitudinal samples were collected from RP (n=25) and SP (n=41) groups at the ages of 3, 6, 12, 18, 24, or ≥ 36 months. We performed a comprehensive metabolomics study, analyzing both polar metabolites and lipids. The sample series included a total of 239 samples for lipidomics and 213 for polar metabolites. Results: We observed that metabolites mediated by gut microbiome, such as those involved in tryptophan metabolism, were the main discriminators between RP and SP. The study identified specific circulating molecules and pathways, including amino acid (threonine), sugar derivatives (hexose), and quinic acid that may define rapid vs. slow progression to type 1 diabetes. However, the circulating lipidome did not appear to play a major role in differentiating between RP and SP. Conclusion/interpretation: Our study suggests that a distinct metabolic profile is linked with the type 1 diabetes progression. The identification of specific metabolites and pathways that differentiate RP from SP may have implications for early intervention strategies to delay the development of type 1 diabetes

HLA-DR-DQ haplotypes and specificity of the initial autoantibody in islet specific autoimmunity

Abstract Objective: We aimed to clarify the association of various HLA risk alleles with different types of autoantibodies initiating islet specific autoimmunity. Methods: Follow-up cohorts from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study and children diagnosed with type 1 diabetes (T1D) from the Finnish Pediatric Diabetes Register (FPDR) were analyzed for the presence of autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), IA-2 antigen (IA-2A), and zinc transporter 8 (ZnT8A); and genotyped for HLA DR/DQ alleles. In the DIPP study, autoantibodies were regularly analyzed from birth up to 15 years of age. Results: In the DIPP cohort, 621 children developed one single persistent autoantibody, GADA in 284, IAA in 268, and IA-2A in 40 cases. Highly significant differences in the specificity of the first autoantibody were observed between HLA genotypes. Homozygotes for the DR3-DQ2 haplotype had almost exclusively GADA as the first autoantibody, whereas a more even distribution between GADA and IAA was found in DR3-DQ2/DR4-DQ8 as well as DR3-DQ/x and DR4-DQ8/x genotypes (x referring to neutral haplotypes). In DR4-DQ8 positive genotypes with the DRB1*04:01 allele IAA was more often the first autoantibody than in DRB1*04:04 positive genotypes. Various neutral haplotypes also significantly affected the relative proportions of different initial autoantibodies. These findings were confirmed and expanded in a series of 1591 T1D children under the age of 10 years from FPDR. Conclusions: These results emphasize the importance of HLA class II polymorphisms in the recognition of autoantigen epitopes in the initiation of various pathways of the autoimmune response

Detection of enterovirus RNA in peripheral blood mononuclear cells correlates with the presence of the predisposing allele of the type 1 diabetes risk gene IFIH1 and with disease stage

Abstract Aims/hypothesis: Enteroviral infection has been implicated consistently as a key environmental factor correlating with the appearance of autoimmunity and/or the presence of overt type 1 diabetes, in which pancreatic insulin-producing beta cells are destroyed by an autoimmune response. Genetic predisposition through variation in the type 1 diabetes risk gene IFIH1 (interferon induced with helicase C domain 1), which encodes the viral pattern-recognition receptor melanoma differentiation-associated protein 5 (MDA5), supports a potential link between enterovirus infection and type 1 diabetes. Methods: We used molecular techniques to detect enterovirus RNA in peripheral blood samples (in separated cellular compartments or plasma) from two cohorts comprising 79 children or 72 adults that include individuals with and without type 1 diabetes who had multiple autoantibodies. We also used immunohistochemistry to detect the enteroviral protein VP1 in the pancreatic islets of post-mortem donors (n=43) with type 1 diabetes. Results: We observed enhanced detection sensitivity when sampling the cellular compartment compared with the non-cellular compartment of peripheral blood (OR 21.69; 95% CI 3.64, 229.20; p&lt;0.0001). In addition, we show that children with autoimmunity are more likely to test positive for enterovirus RNA than those without autoimmunity (OR 11.60; 95% CI 1.89, 126.90; p=0.0065). Furthermore, we found that individuals carrying the predisposing allele (946Thr) of the common variant in IFIH1 (rs1990760, Thr946Ala) are more likely to test positive for enterovirus in peripheral blood (OR 3.07; 95% CI 1.02, 8.58; p=0.045). In contrast, using immunohistochemistry, there was no correlation between the common variant in IFIH1 and detection of enteroviral VP1 protein in the pancreatic islets of donors with type 1 diabetes. Conclusions/interpretation: Our data indicate that, in peripheral blood, antigen-presenting cells are the predominant source of enterovirus infection, and that infection is correlated with disease stage and genetic predisposition, thereby supporting a role for enterovirus infection prior to disease onset