Taina Härkönen

Fecal Microbiota Composition Differs Between Children With β-Cell Autoimmunity and Those Without

The role of the intestinal microbiota as a regulator of autoimmune diabetes in animal models is well-established, but data on human type 1 diabetes are tentative and based on studies including only a few study subjects. To exclude secondary effects of diabetes and HLA risk genotype on gut microbiota, we compared the intestinal microbiota composition in children with at least two diabetes-associated autoantibodies (n = 18) with autoantibody-negative children matched for age, sex, early feeding history, and HLA risk genotype using pyrosequencing. Principal component analysis indicated that a low abundance of lactate-producing and butyrate-producing species was associated with β-cell autoimmunity. In addition, a dearth of the two most dominant Bifidobacterium species, Bifidobacterium adolescentis and Bifidobacterium pseudocatenulatum, and an increased abundance of the Bacteroides genus were observed in the children with β-cell autoimmunity. We did not find increased fecal calprotectin or IgA as marker of inflammation in children with β-cell autoimmunity. Functional studies related to the observed alterations in the gut microbiome are warranted because the low abundance of bifidobacteria and butyrate-producing species could adversely affect the intestinal epithelial barrier function and inflammation, whereas the apparent importance of the Bacteroides genus in development of type 1 diabetes is insufficiently understood.

Natural history of the infant gut microbiome and impact of antibiotic treatment on bacterial strain diversity and stability

A longitudinal strain-level analysis of the infant gut microbiome after repeated antibiotic treatments reveals decreased diversity and stability, as well as transient increases in antibiotic resistance genes. Elucidating the effects of drugs on bugs Despite widespread use of antibiotics in children, the effects of antibiotic exposure on the developing infant gut microbiome have remained underexplored. Here, Yassour et al. present a longitudinal study capturing how the gut microbiome responds to and recovers from antibiotic perturbations. Antibiotic-treated children had less stable and less diverse bacterial communities. Antibiotic resistance genes within the guts of these children peaked after antibiotic treatment but generally returned rapidly to baseline. Delivery mode (vaginal versus cesarean) also had strong long-term effects on microbial diversity. These data give insights into the consequences of early life factors such as birth mode and antibiotic treatment on the infant gut microbiome. The gut microbial community is dynamic during the first 3 years of life, before stabilizing to an adult-like state. However, little is known about the impact of environmental factors on the developing human gut microbiome. We report a longitudinal study of the gut microbiome based on DNA sequence analysis of monthly stool samples and clinical information from 39 children, about half of whom received multiple courses of antibiotics during the first 3 years of life. Whereas the gut microbiome of most children born by vaginal delivery was dominated by Bacteroides species, the four children born by cesarean section and about 20% of vaginally born children lacked Bacteroides in the first 6 to 18 months of life. Longitudinal sampling, coupled with whole-genome shotgun sequencing, allowed detection of strain-level variation as well as the abundance of antibiotic resistance genes. The microbiota of antibiotic-treated children was less diverse in terms of both bacterial species and strains, with some species often dominated by single strains. In addition, we observed short-term composition changes between consecutive samples from children treated with antibiotics. Antibiotic resistance genes carried on microbial chromosomes showed a peak in abundance after antibiotic treatment followed by a sharp decline, whereas some genes carried on mobile elements persisted longer after antibiotic therapy ended. Our results highlight the value of high-density longitudinal sampling studies with high-resolution strain profiling for studying the establishment and response to perturbation of the infant gut microbiome.

Metabolic regulation in progression to autoimmune diabetes

Recent evidence from serum metabolomics indicates that specific metabolic disturbances precede β-cell autoimmunity in humans and can be used to identify those children who subsequently progress to type 1 diabetes. The mechanisms behind these disturbances are unknown. Here we show the specificity of the pre-autoimmune metabolic changes, as indicated by their conservation in a murine model of type 1 diabetes. We performed a study in non-obese prediabetic (NOD) mice which recapitulated the design of the human study and derived the metabolic states from longitudinal lipidomics data. We show that female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, normoglycemia, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum group. Together, the findings indicate that autoimmune diabetes is preceded by a state of increased metabolic demands on the islets resulting in elevated insulin secretion and suggest alternative metabolic related pathways as therapeutic targets to prevent diabetes.

The novel hexapeptide motif found in the acyltransferases LpxA and LpxD of lipid A biosynthesis is conserved in various bacteria.

Two bacterial acyltransferases (LpxA of Escherichia coli, LpxD of E. coli and Salmonella typhimurium) have previously been shown to consist of a very unusual tandem‐repeat structure with tens of repeating hexapeptides (24 hexapeptides in LpxA, 26 in LpxD). By sequencing LpxD of Yersinia enterocolitica (a distant relative of E. coli and S. typhimurium within Enterobacteriaceae) as well as LpxA of S. typhimurium and Y. enterocolitica, and by analyzing the existing data on these enzymes of Ricketsia rickettsii, it was now shown that the hexapeptide repeat pattern is a very conservative property of these enzymes. Even though the overall homology (allowing equivalent amino acids) between the four proteins was only 59% in LpxA and 58% in LpxD, the homology in the first residue of each hexapeptide was 87% in LpxA and 100% in LpxD. Secondary structure prediction by PredictProtein server suggested a very strong beta strand dominance in all the hexad regions. Accordingly, LpxA and LpxD of various bacterial origins can now be regarded as structurally very unusual enzymes, largely consisting of hexad repeats.

Th1/Th17 Plasticity Is a Marker of Advanced β Cell Autoimmunity and Impaired Glucose Tolerance in Humans

Upregulation of IL-17 immunity and detrimental effects of IL-17 on human islets have been implicated in human type 1 diabetes. In animal models, the plasticity of Th1/Th17 cells contributes to the development of autoimmune diabetes. In this study, we demonstrate that the upregulation of the IL-17 pathway and Th1/Th17 plasticity in peripheral blood are markers of advanced β cell autoimmunity and impaired β cell function in human type 1 diabetes. Activated Th17 immunity was observed in the late stage of preclinical diabetes in children with β cell autoimmunity and impaired glucose tolerance, but not in children with early β cell autoimmunity. We found an increased ratio of IFN-γ/IL-17 expression in Th17 cells in children with advanced β cell autoimmunity, which correlated with HbA1c and plasma glucose concentrations in an oral glucose tolerance test, and thus impaired β cell function. Low expression of Helios was seen in Th17 cells, suggesting that Th1/Th17 cells are not converted thymus-derived regulatory T cells. Our results suggest that the development of Th1/Th17 plasticity may serve as a biomarker of disease progression from β cell autoantibody positivity to type 1 diabetes. These data in human type 1 diabetes emphasize the role of Th1/Th17 plasticity as a potential contributor to tissue destruction in autoimmune conditions.

Autoantibodies against zinc transporter 8 are related to age, metabolic state and HLA DR genotype in children with newly diagnosed type 1 diabetes

We set out to define the characteristics of humoral autoimmunity against ZnT8 in children and adolescents with newly diagnosed T1D in relation to age and metabolic status at diagnosis, human leucocyte antigen (HLA) genotype and family history of T1D.

Intestinal virome changes precede autoimmunity in type I diabetes-susceptible children

Significance Type 1 diabetes (T1D) is a major autoimmune disease with increasing incidence in recent years. In this study, we found that the intestinal viromes of cases were less diverse than those of controls. We identified eukaryotic viruses and bacteriophage contigs that are associated with the presence or absence of autoimmunity. These viruses provide targets for future mechanistic studies to differentiate causal and incidental associations between the virome and protection against the development of T1D. Viruses have long been considered potential triggers of autoimmune diseases. Here we defined the intestinal virome from birth to the development of autoimmunity in children at risk for type 1 diabetes (T1D). A total of 220 virus-enriched preparations from serially collected fecal samples from 11 children (cases) who developed serum autoantibodies associated with T1D (of whom five developed clinical T1D) were compared with samples from controls. Intestinal viromes of case subjects were less diverse than those of controls. Among eukaryotic viruses, we identified significant enrichment of Circoviridae-related sequences in samples from controls in comparison with cases. Enterovirus, kobuvirus, parechovirus, parvovirus, and rotavirus sequences were frequently detected but were not associated with autoimmunity. For bacteriophages, we found higher Shannon diversity and richness in controls compared with cases and observed that changes in the intestinal virome over time differed between cases and controls. Using Random Forests analysis, we identified disease-associated viral bacteriophage contigs after subtraction of age-associated contigs. These disease-associated contigs were statistically linked to specific components of the bacterial microbiome. Thus, changes in the intestinal virome preceded autoimmunity in this cohort. Specific components of the virome were both directly and inversely associated with the development of human autoimmune disease.

IA-2 antibody isotypes and epitope specificity during the prediabetic process in children with HLA-conferred susceptibility to type I diabetes

The natural history of preclinical diabetes is partly characterized, but there is still limited information on the dynamics of the immune response to β‐cell autoantigens during the course of preclinical disease. The aim of this work was to assess the maturation of the humoral immune response to the protein tyrosine phosphatase(PTP)‐related proteins (IA‐2 and IA‐2β) in preclinical type I diabetes (TID). Forty‐five children participating in the Finnish Type I Diabetes Prediction and Prevention (DIPP) Study who had seroconverted to IA‐2 antibody positivity were analysed. Specific radiobinding assays were used to determine IA‐2/IA‐2β epitope‐specific antibodies (the juxtamembrane (JM) region of IA‐2, PTP‐like domain and βPTP‐like domain) and isotype‐specific IA‐2 antibodies. Individual areas under the curve (AUC) over the observation period were calculated for total IA‐2 antibodies, each isotype and specific epitope responses. The children who progressed to TID tended to have an initial IA‐2 JM epitope response more frequently (P = 0·06), and this response was more often dominant during the observation period (P < 0·05). The children who did not progress to TID had IgE‐IA‐2 more frequently (70%; versus progressors 27%; P < 0·05), and had higher integrated titres of IgE‐IA‐2 antibodies (P < 0·05). The occurrence of IgE‐IA‐2 antibodies was protective even when combined with positivity for IA‐2 JM antibodies (P = 0·002). IgE‐IA‐2 antibody reactivity may be a marker of a regulatory immune response providing protection against or delaying progression to TID among IA‐2 antibody‐positive young children with HLA‐conferred disease susceptibility.

Characterization of human organ donors testing positive for type 1 diabetes‐associated autoantibodies

In this study we aim to describe the characteristics of non‐diabetic organ donors with circulating diabetes‐associated autoantibodies collected within the Nordic Network for Islet Transplantation. One thousand and thirty organ donors have been screened in Uppsala for antibodies against glutamic acid decarboxylase (GADA) and islet antigen‐2 (IA‐2A). The 32 non‐diabetic donors that tested positive for GADA (3·3% of all non‐diabetic donors) were studied in more detail, together with 32 matched controls. Mean age among the autoantibody‐positive donors was 52·6 (range 21–74), family history of type 1 diabetes (T1D) was unknown, and no donor was genetically predisposed for T1D regarding the human leucocyte antigen (HLA) locus. Subjects were analysed for islet cell antibodies (ICA), insulin autoantibodies (IAA) and zinc transporter 8 antibodies (ZnT8A), and pancreas morphology and clinical data were examined. Eight non‐diabetic donors tested positive for two antibodies and one donor tested positive for four antibodies. No insulitis or other signs of a diabetic process were found in any of the donors. While inflammatory cells were present in all donors, subjects with high GADA titres had significantly higher CD45 cell numbers in exocrine tissue than controls. The extent of fibrosis was more pronounced in autoantibody‐positive donors, even in subjects with lower GADA titres. Notably, it is possible that events not related directly to T1D (e.g. subclinical pancreatitis) may induce autoantibodies in some cases.

Extended Family History of Type 1 Diabetes and Phenotype and Genotype of Newly Diagnosed Children

OBJECTIVE To determine the frequency of newly diagnosed diabetic children with first- and second-degree relatives affected by type 1 diabetes and to characterize the effects of this positive family history on clinical markers, signs of β-cell autoimmunity, and HLA genotype in the index case. RESEARCH DESIGN AND METHODS Children (n = 1,488) with type 1 diabetes diagnosed under 15 years of age were included in a cross-sectional study from the Finnish Pediatric Diabetes Register. Data on family history of diabetes and metabolic decompensation at diagnosis were collected using a questionnaire. Antibodies to β-cell autoantigens (islet cell antibodies, insulin autoantibodies, GAD antibodies, and antibodies to the islet antigen 2 molecule) and HLA genotypes were analyzed. RESULTS A total of 12.2% of the subjects had a first-degree relative with type 1 diabetes (father 6.2%, mother 3.2%, and sibling 4.8%) and 11.9% had an affected second-degree relative. Children without affected relatives had lower pH (P < 0.001), higher plasma glucose (P < 0.001) and β-hydroxybutyrate concentrations (P < 0.001), a higher rate of impaired consciousness (P = 0.02), and greater weight loss (P < 0.001). There were no differences in signs of β-cell autoimmunity. The familial cases carried the HLA DR4-DQ8 haplotype more frequently than sporadic cases (74.0 vs. 67.0%, P = 0.02). CONCLUSIONS When the extended family history of type 1 diabetes is considered, the proportion of sporadic diabetes cases may be reduced to <80%. A positive family history for type 1 diabetes associates with a less severe metabolic decompensation at diagnosis, even when only second-degree relatives are affected. Autoantibody profiles are similar in familial and sporadic type 1 diabetes, suggesting similar pathogenetic mechanisms.