Heli Siljander

The Dynamics of the Human Infant Gut Microbiome in Development and in Progression toward Type 1 Diabetes

Colonization of the fetal and infant gut microbiome results in dynamic changes in diversity, which can impact disease susceptibility. To examine the relationship between human gut microbiome dynamics throughout infancy and type 1 diabetes (T1D), we examined a cohort of 33 infants genetically predisposed to T1D. Modeling trajectories of microbial abundances through infancy revealed a subset of microbial relationships shared across most subjects. Although strain composition of a given species was highly variable between individuals, it was stable within individuals throughout infancy. Metabolic composition and metabolic pathway abundance remained constant across time. A marked drop in alpha-diversity was observed in T1D progressors in the time window between seroconversion and T1D diagnosis, accompanied by spikes in inflammation-favoring organisms, gene functions, and serum and stool metabolites. This work identifies trends in the development of the human infant gut microbiome along with specific alterations that precede T1D onset and distinguish T1D progressors from nonprogressors.

Nasal insulin to prevent type 1 diabetes in children with HLA genotypes and autoantibodies conferring increased risk of disease: a double-blind, randomised controlled trial

BACKGROUND In mouse models of diabetes, prophylactic administration of insulin reduced incidence of the disease. We investigated whether administration of nasal insulin decreased the incidence of type 1 diabetes, in children with HLA genotypes and autoantibodies increasing the risk of the disease. METHODS At three university hospitals in Turku, Oulu, and Tampere (Finland), we analysed cord blood samples of 116 720 consecutively born infants, and 3430 of their siblings, for the HLA-DQB1 susceptibility alleles for type 1 diabetes. 17 397 infants and 1613 siblings had increased genetic risk, of whom 11 225 and 1574, respectively, consented to screening of diabetes-associated autoantibodies at every 3-12 months. In a double-blind trial, we randomly assigned 224 infants and 40 siblings positive for two or more autoantibodies, in consecutive samples, to receive short-acting human insulin (1 unit/kg; n=115 and n=22) or placebo (n=109 and n=18) once a day intranasally. We used a restricted randomisation, stratified by site, with permuted blocks of size two. Primary endpoint was diagnosis of diabetes. Analysis was by intention to treat. The study was terminated early because insulin had no beneficial effect. This study is registered with ClinicalTrials.gov, number NCT00223613. FINDINGS Median duration of the intervention was 1.8 years (range 0-9.7). Diabetes was diagnosed in 49 index children randomised to receive insulin, and in 47 randomised to placebo (hazard ratio [HR] 1.14; 95% CI 0.73-1.77). 42 and 38 of these children, respectively, continued treatment until diagnosis, with yearly rates of diabetes onset of 16.8% (95% CI 11.7-21.9) and 15.3% (10.5-20.2). Seven siblings were diagnosed with diabetes in the insulin group, versus six in the placebo group (HR 1.93; 0.56-6.77). In all randomised children, diabetes was diagnosed in 56 in the insulin group, and 53 in the placebo group (HR 0.98; 0.67-1.43, p=0.91). INTERPRETATION In children with HLA-conferred susceptibility to diabetes, administration of nasal insulin, started soon after detection of autoantibodies, could not be shown to prevent or delay type 1 diabetes.

Autoimmune mechanisms in type 1 diabetes

Type 1 diabetes (T1D) is perceived as a chronic immune-mediated disease with a subclinical prodromal period characterized by selective loss of insulin-producing beta-cells in the pancreatic islets in genetically susceptible subjects. Autoreactive T cells, both CD4 and CD8 cells, have been implicated as active players in beta-cell destruction. A series of autoantigens have been identified in T1D including insulin, glutamic acid decarboxylase (GAD), the protein tyrosine phosphatase-related islet antigen 2 (IA-2), and most recently the zinc transporter Slc30A8 residing in the insulin secretory granule of the beta-cell. The issue whether there is any primary autoantigen in T1D has remained controversial. Given that there are two major HLA haplotypes conferring disease susceptibility, i.e. the DR3-DQ2 haplotype and the DR4-DQ8 haplotype, one may assume that there will be at least two primary antigens in T1D. The first signs of beta-cell autoimmunity might appear already during the first year of life. Autoantibodies may be considered as markers of an ongoing disease process in the pancreatic islets, and they can be used for prediction of T1D in non-diabetic individuals. Autoantigen-specific T-cell responses have been detected from peripheral blood in both patients with T1D and in unaffected subjects, but a clear discrimination between diabetic and non-diabetic subjects have rarely been seen until recently.

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.

The role of the intestinal microbiota in type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) is a chronic immune-mediated disease with a subclinical prodromal period, characterized by selective loss of insulin-producing-β cells in the pancreatic islets of genetically susceptible individuals. The incidence of T1DM has increased several fold in most developed countries since World War II, in conjunction with other immune-mediated diseases. Rapid environmental changes and modern lifestyles are probably the driving factors that underlie this increase. These effects might be mediated by changes in the human microbiota, particularly the intestinal microbiota. Research on the gut microbiome of individuals at risk of developing T1DM and in patients with established disease is still in its infancy, but initial findings indicate that the intestinal microbiome of individuals with prediabetes or diabetes mellitus is different to that of healthy individuals. The gut microbiota in individuals with preclinical T1DM is characterized by Bacteroidetes dominating at the phylum level, a dearth of butyrate-producing bacteria, reduced bacterial and functional diversity and low community stability. However, these changes seem to emerge after the appearance of autoantibodies that are predictive of T1DM, which suggests that the intestinal microbiota might be involved in the progression from β-cell autoimmunity to clinical disease rather than in the initiation of the disease process.

Predictive Characteristics of Diabetes-Associated Autoantibodies Among Children With HLA-Conferred Disease Susceptibility in the General Population

OBJECTIVE As data on the predictive characteristics of diabetes-associated autoantibodies for type 1 diabetes in the general population are scarce, we assessed the predictive performance of islet cell autoantibodies (ICAs) in combination with autoantibodies against insulin (IAAs), autoantibodies against GAD, and/or islet antigen 2 for type 1 diabetes in children with HLA-defined disease predisposition recruited from the general population. RESEARCH DESIGN AND METHODS We observed 7,410 children from birth (median 9.2 years) for β-cell autoimmunity and diabetes. If a child developed ICA positivity or diabetes, the three other antibodies were measured in all samples available from that individual. Persistent autoantibody positivity was defined as continued positivity in at least two sequential samples including the last available sample. RESULTS Pre-diabetic ICA positivity was observed in 1,173 subjects (15.8%), 155 of whom developed type 1 diabetes. With ICA screening, 86% of 180 progressors (median age at diagnosis 5.0 years) were identified. Positivity for four antibodies was associated with the highest disease sensitivity (54.4%) and negative predictive values (98.3%) and the lowest negative likelihood ratio (0.5). The combination of persistent ICA and IAA positivity resulted in the highest positive predictive value (91.7%), positive likelihood ratio (441.8), cumulative disease risk (100%), and specificity (100%). Young age at seroconversion, high ICA level, multipositivity, and persistent positivity for IAA were significant risk markers for type 1 diabetes. CONCLUSIONS Within the general population, the combination of HLA and autoantibody screening resulted in disease risks that are likely to be as high as those reported among autoantibody-positive siblings of children with type 1 diabetes.

ConStrains identifies microbial strains in metagenomic datasets

An important fraction of microbial diversity is harbored in strain individuality, so identification of conspecific bacterial strains is imperative for improved understanding of microbial community functions. Limitations in bioinformatics and sequencing technologies have to date precluded strain identification owing to difficulties in phasing short reads to faithfully recover the original strain-level genotypes, which have highly similar sequences. We present ConStrains, an open-source algorithm that identifies conspecific strains from metagenomic sequence data and reconstructs the phylogeny of these strains in microbial communities. The algorithm uses single-nucleotide polymorphism (SNP) patterns in a set of universal genes to infer within-species structures that represent strains. Applying ConStrains to simulated and host-derived datasets provides insights into microbial community dynamics.

Effect of HLA Class I and Class II Alleles on Progression from Autoantibody Positivity to Overt Type 1 Diabetes in Children with Risk-Associated Class II Genotypes

OBJECTIVE Class II alleles define the main HLA effect on type 1 diabetes, but there is an independent effect of certain class I alleles. Class II and class I molecules are differently involved in the initiation and effector phases of the immune response, suggesting that class I alleles would be important determinants in the rate of β-cell destruction. To test this hypothesis we analyzed the role of HLA class I and class II gene polymorphisms in the progression from diabetes-associated autoimmunity to clinical disease. RESEARCH DESIGN AND METHODS The effect of HLA-DR-DQ haplotypes and a panel of class I HLA-A and -B alleles on the progression from autoantibody seroconversion to clinical diabetes was studied in 249 children persistently positive for at least one biochemical diabetes-associated autoantibody in addition to islet cell autoantibody. RESULTS The progression to clinical disease was separately analyzed after the appearance of the first and the second persistent biochemical autoantibody using Cox regression. Multivariate analysis demonstrated a significant protective effect of the A*03 allele (odds ratio [OR] 0.61, P = 0.042 after the first and OR 0.55, P = 0.027 after the second autoantibody), whereas the B*39 allele had a promoting effect after seroconversion for the second autoantibody (OR 2.4, P = 0.014). When children with the DR3/DR4 genotype were separately analyzed, HLA-B*39 had a strong effect (OR 6.6, P = 0.004 and OR 7.5, P = 0.007, after the appearance of the first and the second autoantibody, respectively). The protective effect of A*03 was seen only among children without the DR3/DR4 combination. CONCLUSIONS These results confirm that class I alleles affect the progression of diabetes-associated autoimmunity and demonstrate interactions between class I and class II alleles.

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.

Microbial Exposure in Infancy and Subsequent Appearance of Type 1 Diabetes Mellitus-Associated Autoantibodies A Cohort Study

IMPORTANCE The role of microbial exposure during early life in the development of type 1 diabetes mellitus is unclear. OBJECTIVE To investigate whether animal contact and other microbial exposures during infancy are associated with the development of preclinical and clinical type 1 diabetes. DESIGN, SETTING, AND PARTICIPANTS A birth cohort of children with HLA antigen-DQB1-conferred susceptibility to type 1 diabetes was examined. Participants included 3143 consecutively born children at 2 hospitals in Finland between 1996 and 2004. EXPOSURES The following exposures during the first year of life were assessed: indoor and outdoor dogs and cats, farm animals, farming, visit to a stable, day care, and exposure to antibiotics during the first week of life. MAIN OUTCOMES AND MEASURES Clinical and preclinical type 1 diabetes were used as outcomes. The latter was defined as repeated positivity for islet-cell antibodies plus for at least 1 of 3 other diabetes-associated autoantibodies analyzed and/or clinical type 1 diabetes. The autoantibodies were analyzed at 3- to 12-month intervals since the birth of the child. RESULTS Children exposed to an indoor dog, compared with otherwise similar children without an indoor dog exposure, had a reduced odds of developing preclinical type 1 diabetes (adjusted odds ratio [OR], 0.47; 95% CI, 0.28-0.80; P = .005) and clinical type 1 diabetes (adjusted OR, 0.40; 95% CI, 0.14-1.14; P = .08). All of the other microbial exposures studied were not associated with preclinical or clinical diabetes: the odds ratios ranged from 0.74 to 1.58. CONCLUSIONS AND RELEVANCE Among the 9 early microbial exposures studied, only the indoor dog exposure during the first year of life was inversely associated with the development of preclinical type 1 diabetes. This finding needs to be confirmed in other populations.