Distinct cellular immune responses in children en route to type 1 diabetes with different first-appearing autoantibodies.

Previous studies have revealed heterogeneity in the progression to clinical type 1 diabetes in children who develop islet-specific antibodies either to insulin (IAA) or glutamic acid decarboxylase (GADA) as the first autoantibodies. Here, we test the hypothesis that children who later develop clinical disease have different early immune responses, depending on the type of the first autoantibody to appear (GADA-first or IAA-first). We use mass cytometry for deep immune profiling of peripheral blood mononuclear cell samples longitudinally collected from children who later progressed to clinical disease (IAA-first, GADA-first, ≥2 autoantibodies first groups) and matched for age, sex, and HLA controls who did not, as part of the Type 1 Diabetes Prediction and Prevention study. We identify differences in immune cell composition of children who later develop disease depending on the type of autoantibodies that appear first. Notably, we observe an increase in CD161 expression in natural killer cells of children with ≥2 autoantibodies and validate this in an independent cohort. The results highlight the importance of endotype-specific analyses and are likely to contribute to our understanding of pathogenic mechanisms underlying type 1 diabetes development.

Urban indoor gardening enhances immune regulation and diversifies skin microbiota - A placebo-controlled double-blinded intervention study.

According to the hygiene and biodiversity hypotheses, frequent exposure to environmental microbiota, especially through soil contact, diversifies commensal microbiota, enhances immune modulation, and ultimately lowers the risk of immune-mediated diseases. Here we test the underlying assumption of the hygiene and biodiversity hypotheses by instructing volunteers to grow edible plants indoors during the winter season when natural exposure to environmental microbiota is low. The one-month randomized, placebo-controlled double-blind trial consisted of two treatments: participants received either microbially diverse growing medium or visually similar but microbially poor growing medium. Skin microbiota and a panel of seven immune markers were analyzed in the beginning of the trial and after one month. The diversity of five bacterial phyla (Bacteroidetes, Planctomycetes, Proteobacteria, Cyanobacteria, and Verrucomicrobia) and one class (Bacteroidia) increased on the skin of participants in the intervention group while no changes were observed in the placebo group. The number of nodes and edges in the co-occurrence networks of the skin bacteria increased on average three times more in the intervention group than in the placebo group. The plasma levels of the immunomodulatory cytokine interleukin 10 (IL-10) increased in the intervention group when compared with the placebo group. A similar trend was observed in the interleukin 17A (IL-17A) levels and in the IL-10:IL-17A ratios. Participants in both groups reported high satisfaction and adherence to the trial. The current study provides evidence in support of the core assumption of the hygiene and biodiversity hypotheses of immune-mediated diseases. Indoor urban gardening offers a meaningful and convenient approach for increasing year-round exposure to environmental microbiota, paving the way for other prophylactic practices that might help prevent immune-mediated diseases.

Coxsackievirus infection induces direct pancreatic ß cell killing but poor antiviral CD8+ T cell responses.

Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß cell autoimmunity and type 1 diabetes. We investigated how CVB affects human ß cells and anti-CVB T cell responses. ß cells were efficiently infected by CVB in vitro, down-regulated human leukocyte antigen (HLA) class I, and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized a fraction of these peptides; only another subfraction was targeted by effector/memory T cells that expressed exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with ß cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Our in vitro and ex vivo data highlight limited CD8+ T cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and nonstructural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

Safety, tolerability and immunogenicity of PRV-101, a multivalent vaccine targeting coxsackie B viruses (CVBs) associated with type 1 diabetes: a double-blind randomised placebo-controlled Phase I trial.

AIMS/HYPOTHESIS: Infection with coxsackie B viruses (CVBs) can cause diseases ranging from mild common cold-type symptoms to severe life-threatening conditions. CVB infections are considered to be prime candidates for environmental triggers of type 1 diabetes. This, together with the significant disease burden of acute CVB infections and their association with chronic diseases other than diabetes, has prompted the development of human CVB vaccines. The current study evaluated the safety and immunogenicity of the first human vaccine designed against CVBs associated with type 1 diabetes in a double-blind randomised placebo-controlled Phase I trial. METHODS: The main eligibility criteria for participants were good general health, age between 18 and 45 years, provision of written informed consent and willingness to comply with all trial procedures. Treatment allocation (PRV-101 or placebo) was based on a computer-generated randomisation schedule and people assessing the outcomes were masked to group assignment. In total, 32 participants (17 men, 15 women) aged 18-44 years were randomised to receive a low (n=12) or high (n=12) dose of a multivalent, formalin-inactivated vaccine including CVB serotypes 1-5 (PRV-101), or placebo (n=8), given by intramuscular injections at weeks 0, 4 and 8 at a single study site in Finland. The participants were followed for another 24 weeks. Safety and tolerability were the primary endpoints. Anti-CVB IgG and virus-neutralising titres were analysed using an ELISA and neutralising plaque reduction assays, respectively. RESULTS: Among the 32 participants (low dose, n=12; high dose, n=12; placebo, n=8) no serious adverse events or adverse events leading to study treatment discontinuation were observed. Treatment-emergent adverse events considered to be related to the study drug occurred in 37.5% of the participants in the placebo group and 62.5% in the PRV-101 group (injection site pain, headache, injection site discomfort and injection site pruritus being most common). PRV-101 induced dose-dependent neutralising antibody responses against all five CVB serotypes included in the vaccine in both the high- and low-dose groups. Protective titres ≥8 against all five serotypes were seen in >90% of participants over the entire follow-up period. CONCLUSIONS/INTERPRETATION: The results indicate that the tested multivalent CVB vaccine is well tolerated and immunogenic, supporting its further clinical development. TRIAL REGISTRATION: ClinicalTrials.gov NCT04690426. FUNDING: This trial was funded by Provention Bio, a Sanofi company.

CXADR polymorphism rs6517774 modifies islet autoimmunity characteristics and exhibits sex disparity.

Enteroviral infections have been linked to the development of islet autoimmunity (IA) and type 1 diabetes (T1D), and the coxsackie and adenovirus receptor (CXADR) is one of the ligands used by adenoviruses and enteroviruses for cell internalization. Two CXADR single nucleotide polymorphisms (SNPs), rs6517774 and rs2824404, were previously associated with an increased susceptibility to IA in the international TEDDY study (The Environmental Determinants of Diabetes in the Young). This study aimed to replicate the results by genotyping 2886 children enrolled in the Finnish Diabetes Prediction and Prevention study (DIPP). In our preliminary analysis of the SNPs' allelic distributions, we could not find any association with IA susceptibility. However, a stratified analysis revealed a sex disparity, since the allelic distribution of rs6517774 was different when comparing autoantibody positive females with males; a difference not seen in healthy subjects. By using HLA risk groups and sex as covariates, a Cox regression survival analysis found that the rs6517774 (A/G) SNP was associated with a lower age at seroconversion in females (Female*rs6517774-AA; HR = 1.53, p = 0.002), while introducing a protective effect in males. Accordingly, we propose that rs6517774 alters IA characteristics by modifying the age at seroconversion in a sex-dependent manner. In light of this observation, rs6517774 now joins a limited set on SNPs found to introduce sex-dependent risk effects on the age at IA initiation.

Distinct transcriptomic profiles in children prior to the appearance of type 1 diabetes-linked islet autoantibodies and following enterovirus infection.

Although the genetic basis and pathogenesis of type 1 diabetes have been studied extensively, how host responses to environmental factors might contribute to autoantibody development remains largely unknown. Here, we use longitudinal blood transcriptome sequencing data to characterize host responses in children within 12 months prior to the appearance of type 1 diabetes-linked islet autoantibodies, as well as matched control children. We report that children who present with insulin-specific autoantibodies first have distinct transcriptional profiles from those who develop GADA autoantibodies first. In particular, gene dosage-driven expression of GSTM1 is associated with GADA autoantibody positivity. Moreover, compared with controls, we observe increased monocyte and decreased B cell proportions 9-12 months prior to autoantibody positivity, especially in children who developed antibodies against insulin first. Lastly, we show that control children present transcriptional signatures consistent with robust immune responses to enterovirus infection, whereas children who later developed islet autoimmunity do not. These findings highlight distinct immune-related transcriptomic differences between case and control children prior to case progression to islet autoimmunity and uncover deficient antiviral response in children who later develop islet autoimmunity.

Pleconaril and ribavirin in new-onset type 1 diabetes: a phase 2 randomized trial.

Previous studies showed a low-grade enterovirus infection in the pancreatic islets of patients with newly diagnosed type 1 diabetes (T1D). In the Diabetes Virus Detection (DiViD) Intervention, a phase 2, placebo-controlled, randomized, parallel group, double-blind trial, 96 children and adolescents (aged 6-15 years) with new-onset T1D received antiviral treatment with pleconaril and ribavirin (n = 47) or placebo (n = 49) for 6 months, with the aim of preserving ß cell function. The primary endpoint was the mean stimulated C-peptide area under the curve (AUC) 12 months after the initiation of treatment (less than 3 weeks after diagnosis) using a mixed linear model. The model used longitudinal log-transformed serum C-peptide AUCs at baseline, at 3 months, 6 months and 1 year. The primary endpoint was met with the serum C-peptide AUC being higher in the pleconaril and ribavirin treatment group compared to the placebo group at 12 months (average marginal effect = 0.057 in the linear mixed model; 95% confidence interval = 0.004-0.11, P = 0.037). The treatment was well tolerated. The results show that antiviral treatment may preserve residual insulin production in children and adolescent with new-onset T1D. This provides a rationale for further evaluating antiviral strategies in the prevention and treatment of T1D. European Union Drug Regulating Authorities Clinical Trials identifier: 2015-003350-41 .

Coxsackievirus infection induces direct pancreatic ß-cell killing but poor anti-viral CD8+ T-cell responses.

Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß-cell autoimmunity. We investigated how CVB impacts human ß cells and anti-CVB T-cell responses. ß cells were efficiently infected by CVB in vitro, downregulated HLA Class I and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized only a fraction of these peptides, and only another sub-fraction was targeted by effector/memory T cells that expressed the exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with the ß-cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Thus, our in-vitro and ex-vivo data highlight limited T-cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and non-structural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

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

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.

The edible plant microbiome: evidence for the occurrence of fruit and vegetable bacteria in the human gut.

Diversity of the gut microbiota is crucial for human health. However, whether fruit and vegetable associated bacteria contribute to overall gut bacterial diversity is still unknown. We reconstructed metagenome-assembled genomes from 156 fruit and vegetable metagenomes to investigate the prevalence of associated bacteria in 2,426 publicly available gut metagenomes. The microbiomes of fresh fruits and vegetables and the human gut are represented by members in common such as Enterobacterales, Burkholderiales, and Lactobacillales. Exposure to bacteria via fruit and vegetable consumption potentially has a beneficial impact on the functional diversity of gut microbiota particularly due to the presence of putative health-promoting genes for the production of vitamin and short-chain fatty acids. In the human gut, they were consistently present, although at a low abundance, approx. 2.2%. Host age, vegetable consumption frequency, and the diversity of plants consumed were drivers favoring a higher proportion. Overall, these results provide one of the primary links between the human microbiome and the environmental microbiome. This study revealed evidence that fruit and vegetable-derived microbes could be found in the human gut and contribute to gut microbiome diversity.

Serum APOC1 levels are decreased in young autoantibody positive children who rapidly progress to type 1 diabetes.

Better understanding of the early events in the development of type 1 diabetes is needed to improve prediction and monitoring of the disease progression during the substantially heterogeneous presymptomatic period of the beta cell damaging process. To address this concern, we used mass spectrometry-based proteomics to analyse longitudinal pre-onset plasma sample series from children positive for multiple islet autoantibodies who had rapidly progressed to type 1 diabetes before 4 years of age (n = 10) and compared these with similar measurements from matched children who were either positive for a single autoantibody (n = 10) or autoantibody negative (n = 10). Following statistical analysis of the longitudinal data, targeted serum proteomics was used to verify 11 proteins putatively associated with the disease development in a similar yet independent and larger cohort of children who progressed to the disease within 5 years of age (n = 31) and matched autoantibody negative children (n = 31). These data reiterated extensive age-related trends for protein levels in young children. Further, these analyses demonstrated that the serum levels of two peptides unique for apolipoprotein C1 (APOC1) were decreased after the appearance of the first islet autoantibody and remained relatively less abundant in children who progressed to type 1 diabetes, in comparison to autoantibody negative children.

Comparison of the effect of autoclaved and non-autoclaved live soil exposure on the mouse immune system : Effect of soil exposure on immune system.

BACKGROUND: . Lack of exposure to the natural microbial diversity of the environment has been linked to dysregulation of the immune system and numerous noncommunicable diseases, such as allergies and autoimmune disorders. Our previous studies suggest that contact with soil material, rich in naturally occurring microbes, could have a beneficial immunoregulatory impact on the immune system in mice and humans. However, differences in the immunomodulatory properties of autoclaved, sterile soil material and non-autoclaved, live soil material have not been compared earlier. RESULTS: . In this study, we exposed C57BL/6 mice to autoclaved and live soil powders that had the same rich microbiota before autoclaving. We studied the effect of the soil powders on the mouse immune system by analyzing different immune cell populations, gene expression in the gut, mesenteric lymph nodes and lung, and serum cytokines. Both autoclaved and live soil exposure were associated with changes in the immune system. The exposure to autoclaved soil resulted in higher levels of Rorγt, Inos and Foxp3 expression in the colon. The exposure to live soil was associated with elevated IFN-γ concentration in the serum. In the mesenteric lymph node, exposure to live soil reduced Gata3 and Foxp3 expression, increased the percentage of CD8 + T cells and the expression of activation marker CD80 in XCR1+SIRPα- migratory conventional dendritic cell 1 subset. CONCLUSIONS: . Our results indicate that exposure to the live and autoclaved soil powders is not toxic for mice. Exposure to live soil powder slightly skews the immune system towards type 1 direction which might be beneficial for inhibiting type 2-related inflammation. Further studies are warranted to quantify the impact of this exposure in experimental type 2 inflammation.

Human intestinal organoid models for celiac disease research.

Celiac disease pathogenesis, in addition to immune cell component, encompasses pathogenic events also in the duodenal epithelium. In celiac disease patients, exposure to dietary gluten induces drastic changes in epithelial differentiation and elicit cellular response to inflammatory cytokines. The autoantigen in celiac disease, transglutaminase 2 (TG2) enzyme, has been also suggested to play its pathogenic gliadin deamidation event in the intestinal epithelium. Therefore in vitro epithelial cell-line models have been exploited in the past to study these pathogenic mechanisms, but they are hampered by their simplistic nature lacking proper cell-type composition and intestinal environ. Moreover, these cell models harbor many cancer-related mutations in tumor suppressor genes making them unsuitable for studying cell differentiation. Intestinal organoids provide a near-native epithelial cell model to study pathogenic agents and mechanisms related to celiac disease. Here we describe protocols to initiate and maintain celiac patient-derived organoid cultures and how to grow them in alternative ways allowing their exploitation in different kind of experiments.

Gastrointestinal Infections Modulate the Risk for Insulin Autoantibodies as the First-Appearing Autoantibody in the TEDDY Study.

OBJECTIVE: To investigate gastrointestinal infection episodes (GIEs) in relation to the appearance of islet autoantibodies in The Environmental Determinants of Diabetes in the Young (TEDDY) cohort. RESEARCH DESIGN AND METHODS: GIEs on risk of autoantibodies against either insulin (IAA) or GAD (GADA) as the first-appearing autoantibody were assessed in a 10-year follow-up of 7,867 children. Stool virome was characterized in a nested case-control study. RESULTS: GIE reports (odds ratio [OR] 2.17 [95% CI 1.39-3.39]) as well as Norwalk viruses found in stool (OR 5.69 [1.36-23.7]) at <1 year of age were associated with an increased IAA risk at 2-4 years of age. GIEs reported at age 1 to <2 years correlated with a lower risk of IAA up to 10 years of age (OR 0.48 [0.35-0.68]). GIE reports at any other age were associated with an increase in IAA risk (OR 2.04 for IAA when GIE was observed 12-23 months prior [1.41-2.96]). Impacts on GADA risk were limited to GIEs <6 months prior to autoantibody development in children <4 years of age (OR 2.16 [1.54-3.02]). CONCLUSIONS: Bidirectional associations were observed. GIEs were associated with increased IAA risk when reported before 1 year of age or 12-23 months prior to IAA. Norwalk virus was identified as one possible candidate factor. GIEs reported during the 2nd year of life were associated with a decreased IAA risk.

Vemurafenib Inhibits Acute and Chronic Enterovirus Infection by Affecting Cellular Kinase Phosphatidylinositol 4-Kinase Type IIIß.

Enteroviruses are one of the most abundant viruses causing mild to serious acute infections in humans and also contributing to chronic diseases like type 1 diabetes. Presently, there are no approved antiviral drugs against enteroviruses. Here, we studied the potency of vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, as an antiviral against enteroviruses. We showed that vemurafenib prevented enterovirus translation and replication at low micromolar dosage in an RAF/MEK/ERK-independent manner. Vemurafenib was effective against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect was related to a cellular phosphatidylinositol 4-kinase type IIIß (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevented infection efficiently in acute cell models, eradicated infection in a chronic cell model, and lowered virus amounts in pancreas and heart in an acute mouse model. Altogether, instead of acting through the RAF/MEK/ERK pathway, vemurafenib affects the cellular PI4KB and, hence, enterovirus replication, opening new possibilities to evaluate further the potential of vemurafenib as a repurposed drug in clinical care. IMPORTANCE Despite the prevalence and medical threat of enteroviruses, presently, there are no antivirals against them. Here, we show that vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, prevents enterovirus translation and replication. Vemurafenib shows efficacy against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect acts through cellular phosphatidylinositol 4-kinase type IIIß (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevents infection efficiently in acute cell models, eradicates infection in a chronic cell model, and lowers virus amounts in pancreas and heart in an acute mouse model. Our findings open new possibilities to develop drugs against enteroviruses and give hope for repurposing vemurafenib as an antiviral drug against enteroviruses.

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

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.

Skin, gut, and sand metagenomic data on placebo-controlled sandbox biodiversity intervention study.

The metagenomic data presented in this article are related to the published research of "A Placebo-controlled double-blinded test of the biodiversity hypothesis of immune-mediated diseases: Environmental microbial diversity elicits changes in cytokines and increase in T regulatory cells in young children" This database contains 16S ribosomal RNA (rRNA) metagenomics of sandbox sand and skin and gut microbiota of children in the intervention and placebo daycares. In intervention daycares, children aged 3-5 years were exposed to playground sand enriched with microbially diverse soil. In placebo daycares, children were exposed to visually similar as in intervention daycares, but microbially poor sand colored with peat. Sand, skin and gut metagenomics were analyzed at baseline and after 14 and 28 days of intervention by high throughput sequencing of bacterial 16S rRNA gene on the Illumina MiSeq platform. This dataset shows how skin bacterial community composition, including classes Gammaproteobacteria and Bacilli, changed, and how the relative abundance of over 30 bacterial genera shifted on the skin of children in the intervention treatment, while no shifts occurred in the placebo group.

Respiratory infections and type 1 diabetes: Potential roles in pathogenesis.

Among the environmental factors associated with type 1 diabetes (T1D), viral infections of the gut and pancreas has been investigated most intensely, identifying enterovirus infections as the prime candidate trigger of islet autoimmunity (IA) and T1D development. However, the association between respiratory tract infections (RTI) and IA/T1D is comparatively less known. While there are significant amounts of epidemiological evidence supporting the role of respiratory infections in T1D, there remains a paucity of data characterising infectious agents at the molecular level. This gap in the literature precludes the identification of the specific infectious agents driving the association between RTI and T1D. Furthermore, the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on the development of IA/T1D remains undeciphered. Here, we provide a comprehensive overview of the evidence to date, implicating RTIs (viral and non-viral) as potential risk factors for IA/T1D.