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

Fecal Microbiota Composition Differs Between Children With β-Cell Autoimmunity and Those Without ORIGINAL ARTICLE Fecal Microbiota Composition Differs Between Children With b-Cell Autoimmunity and Those Without 1 2 3,4,5 6,7 2 Marcus C. de Goffau, Kristiina Luopajärvi, Mikael Knip, Jorma Ilonen, Terhi Ruohtula, 3 2 2 1 1 Taina Härkönen, Laura Orivuori, Saara Hakala, Gjalt W. Welling, Hermie J. Harmsen, and Outi Vaarala role of the gut immune system in the pathogenesis of T1D The role of the intestinal microbiota as a regulator of autoim- has been supported by studies showing an immunological mune diabetes in animal models is well-established, but data on link between the pancreas and the gastrointestinal tract. It human type 1 diabetes are tentative and based on studies including only a few study subjects. To exclude secondary effects has been demonstrated that oral antigens are capable of of diabetes and HLA risk genotype on gut microbiota, we activating antigen-specific T cells in pancreatic lymph compared the intestinal microbiota composition in children with nodes (6) and that the interaction between endothelium at least two diabetes-associated autoantibodies (n = 18) with and T cells is controlled by shared homing receptors in autoantibody-negative children matched for age, sex, early feed- inflamed islets and in the gut (7). The development of ing history, and HLA risk genotype using pyrosequencing. Princi- autoimmune diabetes in animal models is regulated by pal component analysis indicated that a low abundance of factors affecting the function of the gut immune system, lactate-producing and butyrate-producing species was associated such as dietary factors and microbial stimuli, which fur- with b-cell autoimmunity. In addition, a dearth of the two most ther affect the intestinal mucosal barrier and immune re- dominant Bifidobacterium species, Bifidobacterium adolescentis and Bifidobacterium pseudocatenulatum, and an increased sponsiveness (8). The effects of intestinal microbes may abundance of the Bacteroides genus were observed in the chil- not be restricted to barrier mechanisms, but gut micro- dren with b-cell autoimmunity. We did not find increased fecal biota seems to play a key role in the regulation of the T-cell calprotectin or IgA as marker of inflammation in children with populations in the gut, including regulatory T cells, T b-cell autoimmunity. Functional studies related to the observed helper 1, and T helper 17 cells (9). alterations in the gut microbiome are warranted because the low Several animal studies indicate that alterations in the abundance of bifidobacteria and butyrate-producing species intestinal microbiota are associated with the development could adversely affect the intestinal epithelial barrier function of autoimmune diabetes. Nonobese diabetic mice lacking and inflammation, whereas the apparent importance of the Bac- MyD88, an essential signal transducer in Toll-like receptor teroides genus in development of type 1 diabetes is insufficiently signaling, did not have development of diabetes (10), understood. Diabetes 62:1238–1244, 2013 which emphasizes the role of intestinal microbiota as a regulator of autoimmune diabetes. There are differences in the gut microbiota between bio-breeding (BB) diabetes- ype 1 diabetes (T1D) is caused by the destruction prone (DP), and diabetes-resistant rats before the diagnosis of the pancreatic b-cells in genetically suscepti- of diabetes. Antibiotics also can prevent autoimmune di- ble individuals. The disease is considered to be abetes in BB-DP rats (11). Furthermore, it has been Timmune mediated, and the appearance of circu- reported that stool from BB diabetes-resistant rats con- lating autoantibodies against b-cells is seen years before tained more probiotic-like bacteria, whereas Bacteroides, the diagnosis along with a significant reduction in b-cell Eubacterium, and Ruminococcus were more prevalent in mass (1,2). Environmental factors associated with the ac- BB-DP rats (12). Lactobacillus johnsonii prevented di- tivation of the gut immune system, such as early exposure abetes when administered to BB-DP rats (13). There are to dietary antigens (cow’s milk and gluten), have been only a few studies of the intestinal microbiota in relation to associated with the induction of this process (3–5). The T1D in humans, but the results of a follow-up study in- cluding four children with development of T1D suggested that the Bacteroidetes-to-Firmicutes ratio increased over From the Department of Medical Microbiology, University Medical Center time in those children with eventual progression to clinical Groningen and University of Groningen, Groningen, the Netherlands; the T1D, whereas it decreased in children who remained Immune Response Unit, Department of Vaccination and Immune Protec- nondiabetic (14). tion, National Institute for Health and Welfare, Helsinki, Finland; the Children’s Hospital, University of Helsinki and Helsinki University Central The aim of this study was to compare the composition of Hospital, Helsinki, Finland; the Folkhälsan Research Center, Helsinki, Fin- the gut microbiota between children with b-cell autoim- land; the Department of Pediatrics, Tampere University Hospital, Tampere, munity and autoantibody-negative children matched for Finland; the Immunogenetics Laboratory, University of Turku, Turku, Finland; and the Department of Clinical Immunology, University of Eastern age, sex, HLA risk genotype, and early feeding history Finland, Kuopio, Finland. using pyrosequencing as the method of choice. Corresponding author: Outi Vaarala, outi.vaarala@thl.fi. Received 2 May 2012 and accepted 2 November 2012. DOI: 10.2337/db12-0526. Clinical trial reg. nos. NCT00570102 and NCT01055080, clinicaltrials.gov. RESEARCH DESIGN AND METHODS This article contains Supplementary Data online at http://diabetes The current study included 18 children with HLA-conferred susceptibility to .diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0526/-/DC1. T1D who had development of signs of progressive b-cell autoimmunity, i.e., M.C.d.G. and K.L. contributed equally to this study. tested positive for at least two diabetes-associated autoantibodies (cases). 2013 by the American Diabetes Association. Readers may use this article as Eighteen control children were matched for age, sex, and HLA-DQB1 geno- long as the work is properly cited, the use is educational and not for profit, type, as well as for the time of exposure to and the type of infant formula. The and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. characteristics of the children recruited to the gut microbiota study are shown 1238 DIABETES, VOL. 62, APRIL 2013 diabetes.diabetesjournals.org M.C. DE GOFFAU AND ASSOCIATES in Table 1 and are shown in detail in Supplementary Table 1. The study sub- positive for three autoantibodies, and five were positive for two autoanti- jects were recruited from the study population of two intervention trials bodies (Table 1). performed in Finland: the Trial to Reduce IDDM in the Genetically at Risk HLA genotyping. HLA genotyping was performed according to the screening (TRIGR) pilot (n = 20) or the Finnish Dietary Intervention Trial for Prevention protocol in the TRIGR and FINDIA studies. The initial HLA-DQB1 typing for of Type 1 Diabetes (FINDIA) pilot study (n = 16) (3,15). In the TRIGR pilot risk-associated (DQB1*02, DQB1*03:02) and protective (DQB1*03:01, study, autoantibody positivity was monitored until the age of 10 years and in DQB1*06:02, and DQB1*06:03) alleles was complemented with DQA1 typing the ongoing FINDIA pilot study and follow-up time for autoantibodies varied for DQA1*02:01 and DQA1*05 alleles in those with DQB1*02 without pro- from 3 to 6 years. Eighteen of 26 children who had development of at least two tective alleles or the major risk allele DQB1*03:02. This two-step screening autoantibodies in these intervention studies but who had not yet had pro- technique is based on the hybridization of PCR products with lanthanide- gression to overt T1D participated in the current study. The fecal samples labeled probes detected by time-resolved fluorometry as described previously from the study subjects recruited from the FINDIA and TRIGR studies were (17,18). similarly collected between March 2009 and February 2010. Fecal samples DNA extraction. Total DNA was extracted from 0.25 g fecal sample using the from children were collected using stool collection vials and immediately repeated bead beating method described in detail by Yu et al. (19), with stored in home freezers (220°C). Families delivered the frozen sample to the a number of modifications. In brief, four 3-mm glass beads were added during study center, and the sample was stored at 280°C until processing. The fecal the homogenization step, whereas 0.5-mm glass beads were not used at all. samples were collected at a point in time when the study subjects did not have Bead beating was performed using a Precellys 24 (Bertin Technologies, gastroenteritis and had not received any antibiotic treatment during the past 3 Montigny le Bretonneux, France) at 5.5 ms in three rounds of 1 min each months. Four children had developed T1D after the fecal samples were col- with 30-s pauses at room temperature in between. The incubation temperature lected. The control children remained negative for T1D and for all four after the bead beating was increased from 70°C to 95°C. Importantly, protein autoantibodies analyzed. The study was approved by the ethics committees of precipitation with 260 mL ammonium acetate was performed twice. Elution of the participating hospitals and the families gave their written informed con- DNA from the purification columns was performed twice. Columns from the sent. QiaAmp Stool kit were replaced by those from the QIAamp DNA Mini Kit In the TRIGR pilot study, infants with a first-degree relative affected by T1D (Qiagen, Hilden, Germany). were randomized to receive either a regular cow’s milk formula (Enfamil; Pyrosequencing. From each sample, the 16S rRNA genes were amplified using Mead Johnson, Evansville, IN) or an extensively hydrolyzed casein-based test a primer set corresponding to primers 27F-degS (20) and 534-R (21). These formula (Nutramigen; Mead Johnson) until the age of 6–8 months. In the PCR primers target the V1, V2, and V3 hypervariable regions of the 16 S rRNA; FINDIA study, infants were randomized to receive a standard cow’s milk– 27-degS was chosen in particular because it appears to provide a more com- based formula (Tutteli; Valio, Helsinki, Finland), a whey-based hydrolyzed plete assessment of actinobacterial abundance (20). Pyrosequencing was formula (Peptidi-Tutteli), or a whey-based FINDIA formula from which bo- performed using a Roche FLX Genome Sequencer at DNAvision (Liège, Bel- vine insulin was removed. In both studies exclusive breastfeeding was en- gium) using their standard protocol (22). couraged. Sequencing quality control. Pyrosequencing produced a total of 461,874 Autoantibody assays. Insulin autoantibodies, autoantibodies against the 65- reads of 16S rDNA. Sequences were assigned to samples according to sample- kDa isoform of glutamic acid decarboxylase, and autoantibodies against the specific barcodes. Using the Galaxy Tools web site (23), SFF files from the 454 protein tyrosinase phosphatase–related IA-2 molecule (IA-2A) were mea- Genome Sequencer FLX were converted into FASTA files and FASTA quality sured by specific radiobinding assays, and islet cell antibodies were mea- files. FASTA formatted files contained an average (6 SD) of 12,830 6 4,888 sured by a standard immunofluorescence assay as described previously (16). reads per sample. The RDP pyrosequencing pipeline (24) (RDP 10 database, Six out of 18 cases tested positive for four autoantibodies, seven were update 17) was subsequently used to check the FASTA sequence files for the same criteria as described by De Filippo et al. (22) and to check that the av- erage experimental quality score was at least 20. After this quality check, the FASTA files contained an average (6 SD) of 8,024 6 3,136 high-quality reads. TABLE 1 Classification. Taxonomy (phylum, family, and genus level) was assigned Characteristics of the study subjects using RDP classifier 2.01 (25). Richness and diversity analyses were performed as described by De Filippo et al. (22). Identification to the species level was Case children Control children performed using ARB software (26). For this, SSU reference database Characteristics (n) (n = 18) (n = 18) (SSURef_106_ SILVA_19_03_11) was downloaded from the SILVA web site (27). From this database, only sequences of cultured and identified isolates Female/male 7/11 7/11 were used. From these sequences, a “PT-server” database was built, which T1D in first-degree was subsequently used to find the closest match for each of our high-quality relative 10 10 sequences imported from the FASTA files. For this, the “search next relatives Age (years) of listed species in PT-server” function was applied with the following settings: TRIGR pilot study 13.3 (11.7214.2) 12.8 (11.9213.6) oligo length, 12; mismatches, 0; match score, relative; and minimum score, 10. FINDIA pilot study 5.1 (4.926.0) 5.0 (3.9–7.0) The average (6 SD) match score was 75.2 6 18.5. Sequences that were HLA-DQB1 genotype identified as being from different strains but belonging to the same species were grouped together. Species that represented .0.005% of all sequences *02/03:02 7 7 were taken along for statistical analysis, together representing 99.2 6 0.35% of *03:02/x 8 8 all high-quality FASTA reads per sample. *02(DQA1*05)/y 2 2 Assay of IgA and calprotectin in stool samples. A beaker in the bottom cap *02(DQA1*02:01) 1 1 of the extraction device was filled with thawed and homogenized stool sample, Study formula avoiding seeds and grains (;100 mg). The extraction tube was filled with 4.9 CM 10 10 mL extraction buffer and vortexed for 30 s. Mixing was continued in a shaker HC 4 4 at 1,000 rpm for 3 min. The particles were allowed to settle before 10-min HW 3 3 centrifugation at 10,000g at room temperature. Supernatant was collected and FINDIA 1 1 stored at 220°C. Duration of exclusive IgA values were measured with the modified ELISA method described by BF (mo) 2.9 (025.5) 4.0 (0.126.0) Lehtonen et al. (28). Calprotectin levels in stool samples were determined using Calprolab calprotectin ELISA tests (Lot CALP-Pilot3; Calpro AS, Total duration of BF Lysaker, Norway); 20 mL extract was mixed with 980 mL of sample dilution (mo) 8.1 (1.6–16.5) 10.5 (5.1–19.3), P = 0.03 buffer. Cesarean delivery 4 3 Statistical analysis. Principal component analysis (PCA) was performed to find clusters of similar groups of samples or species. PCA is an ordination Cases are children positive for at least two diabetes-associated auto- method based on multivariate statistical analysis that maps the samples in antibodies and control subjects are negative for b-cell autoantibod- ies. The study subjects were participants in the TRIGR and FINDIA different dimensions. All tests were performed with PASW Statistics 18 (SPSS, pilot studies. Data are number or medians (with range). x  *03:01 or Chicago, IL). Initial analysis of the samples showed that bacterial populations *06:02, y  *03:01, *06:02, or *06:03. BF, breastfeeding; CM, cow’s were most often not normally distributed. Mann-Whitney U and Spearman r milk formula; FINDIA, insulin-free whey-based formula; HC, hydro- and x tests were used. All tests were two-tailed. P , 0.05 was considered to lyzed casein-based formula; HW, hydrolyzed whey-based formula. indicate statistical significance. diabetes.diabetesjournals.org DIABETES, VOL. 62, APRIL 2013 1239 FECAL MICROBIOTA COMPOSITION RESULTS important observation illustrated in Fig. 2. PC2 showed an inverse correlation with the abundance of B. adolescentis, Comparison of sequence diversity at phylum, family, but a positive correlation with the abundance of Bifido- and genus levels. The analysis of the high-quality reads of bacterium pseudocatenulatum (P =1 3 10 and P =1 3 all samples at the phylum level showed that Firmicutes 10 ; Spearman r test). B. adolescentis and B. pseudoca- (58.1%), Actinobacteria (36.2%), and Bacteroidetes (3.4%) tenulatum represented the two most commonly identified were the most dominant phyla. On the family level, the species (11.0 and 9.1%, respectively). The PC2 score dif- Bifidobacteriaceae (32.8%; Actinobacteria), the Lachnospir- fered significantly between the children from the TRIGR aceae (18.4%; Firmicutes), and Ruminococcaceae (17.1%; study representing older children and the FINDIA children Firmicutes) were the most common. On the genus level, who were younger (P = 0.001; Mann Whitney U test). the Bifidobacterium genus was the most frequent B. adolescentis was the most common species (15.8%) (34.2%). The most interesting finding was that the Bac- among the children in the TRIGR pilot study, and their teroidetes phylum, the Bacteroidaceae family (2.5%), and samples were clustered in the left leg of the chevron-like the Bacteroides genus (3.1%) were more common in autoantibody-positive children than in autoantibody- distribution, whereas B. pseudocatenulatum was most frequent (15.8%) in the children from the FINDIA study, negative peers (4.6 vs. 2.2%, 3.5 vs. 1.5%, and 4.3 vs. 2.0%, respectively; P = 0.035, 0.022, and 0.031, respectively; and their samples were clustered in the right leg. PC3 was inversely related to the abundance of both B. adolescentis Mann-Whitney U test). Species level PCA. PCA analysis of the species level and B. pseudocatenulatum (P = 0.023 and P = 0.002; Spearman r test), and it is characterized by the sum of revealed various correlations with b-cell autoimmunity. The first principal component (PC1) (46.0%) correlated B. adolescentis and B. pseudocatenulatum (P =1 3 10 ; Spearman r test). Various bacteria were inversely associ- positively with a number of important short-chain fatty acid–producing species (P # 0.01 for all species; Spear- ated with the combined count of these two bifidobacteria, especially the members of the Clostridium cluster XI (P = man r test), such as Bifidobacterium adolescentis (11%), Faecalibacterium prausnitzii (5.6%), Clostridium clos- 6 3 10 ).The apex, encompassed by a circle in Fig. 2, can be described as comprising those samples in which the tridioforme (2.3%), and Roseburia faecis (0.94%), as shown in Fig. 1. This PC1 was inversely related to the combined abundance of B. adolescentis and B. pseudo- catenulatum is ,12%. The children with b-cell autoim- number of diabetes-associated autoantibodies in children (Fig. 1; P = 0.018; Spearman r test). The children with four munity were over-represented in the apex when compared with control children (10/18 vs. 4/18; P = 0.040; x test). autoantibodies had a significantly lower PC1 score, i.e., they had lower numbers of short-chain fatty acid pro- Species-level analysis. The association of single bacte- rial species with autoantibody positivity is shown in ducers than the control children (P = 0.008; Mann-Whitney U test). PC1 score was especially lower in IA-2A–positive Table 2. Roseburia faecis (0.94%) was more abundant in autoantibody-negative than autoantibody-positive children children than in children negative for IA-2A (P = 0.004; Mann-Whitney U test). (P = 0.009; Mann-Whitney U test), whereas Clostridium per- A remarkable chevron-like distribution of dots in the fringens (0.03%) were more abundant in children with b-cell second PC (PC2) and third PC (PC3) provided another autoimmunity than in those without (P = 0.18; Mann-Whitney FIG. 1. The association of PC1 with autoantibody positivity was demonstrated as an inverse correlation between PC1 and number of b-cell autoantibodies in the study cohort (P = 0.018; Spearman r test) and as a difference in PC1 score between the children positive for four auto- antibodies and control children negative for autoantibodies (P = 0.008; Mann-Whitney test). Control children are indicated by being positive for no autoantibodies and cases are positive for two, three, or four autoantibodies (x-axis). Correlations between PC1 and bacterial groups are shown in right panel. 1240 DIABETES, VOL. 62, APRIL 2013 diabetes.diabetesjournals.org M.C. DE GOFFAU AND ASSOCIATES FIG. 2. The distribution of children positive for b-cell autoantibodies (case subjects with open symbols) and autoantibody-negative children (control subjects with filled symbols) according to PC2 (x-axis) and PC3 (y-axis). PC2 shows an inverse correlation with the abundance of B. adolescentis and a positive correlation with B. pseudocatenulatum, whereas PC3 shows an inverse correlation with both B. adolescentis and B. pseudocatenulatum. Indicated percentages indicate the prevalence of B. adolescentis and B. pseudocatenulatum, respectively. Age of the chil- dren is associated with PC2, whereas autoantibody positivity is associated with PC3. The children 3 to 7 years of age (circle) from the FINDIA pilot have higher numbers of B. pseudocatenulatum and lower numbers of B. adolescentis than the children from the TRIGR pilot 11 to 14 years of age (square). The main correlations with PC2 and PC3 are depicted with vectors in the top left. Children with a dearth of both B. adolescentis and B. pseudocatenulatum (sum <12%) are encompassed by the dashed circle near the top and the number of autoantibody-positive children is higher than the number of control subjects at the apex (10/18 vs. 4/18; P = 0.040; x test). U test). The Bacteroides genus was associated with auto- In children from the FINDIA study, it is furthermore antibody positivity, but only a few of its many members noteworthy that Eubacterium hallii (6.0%) was strongly were observed to be related on the species level. In- inversely related to the number of b-cell autoantibodies terestingly, the correlation of the Bacteroides genus with (P = 0.002; Spearman r correlation test). autoantibody positivity was significant only in males, not IgA and calprotectin levels. Calprotectin levels, a in females (P = 0.005 vs. 0.655, respectively). marker of intestinal inflammation, were not observed to be correlated with autoantibody positivity. Clostridium orbiscindens (0.47%) correlated inversely with calpro- TABLE 2 tectin levels (P = 0.004; Spearman r test). Lower levels of Association between single bacterial species and signs of b-cell calprotectin were seen in children with the lower-risk HLA autoimmunity genotype, i.e., (DR3)DQB1*02-DQA1*05, when compared with the children with moderate-risk or high-risk HLA risk Mann-Whitney Variable % U test genotypes (P = 0.001 and 0.005; Mann-Whitney U test). Fecal IgA levels did not differ between autoantibody- *Roseburia faecis (2) 0.94 0.009 positive and autoantibody-negative children but were in- *Clostridium xylanovorans (2) 0.03 0.031 versely correlated with age (P = 0.007; Mann-Whitney U *Peptoniphilus gorbachii (2) 0.006 0.008 test). *Eubacterium hallii (2) 6.0 0.088 Diversity analyses. The analysis of the 90, 95, and 97% *Eubacterium desmolans (2) 0.15 0.058 operational taxonomic similarity levels showed that the *Acetanaerobacterium elongatum (2) 0.06 0.043 diversity was significantly higher in the children in the Bifidobacterium animalis (+) 0.18 0.028 Lactobacillus acidophilus (+) 0.03 0.037 TRIGR pilot cohort than in the children in the FINDIA pilot Clostridium perfringens (+) 0.03 0.018 cohort (P , 0.001; Mann-Whitney U test). Furthermore, Bacteroides genus (+) 3.1 0.031 there was a trend that the diversity per sample was higher Bacteroides genus males (+) 3.6 0.005 in autoantibody-negative children than in the autoantibody- Bacteroides genus females (+) 2.3 0.655 positive children (Supplementary Table 2). This difference B. vulgatus males (+) 1.3 0.023 in diversity, measured in the number of observed opera- B. ovatus males (+) 0.21 0.049 tional taxonomic units and the Chao index, was signifi- B. fragilis females (+) 0.16 0.017 cantly different at the 95% taxonomic similarity level when B. thetaiota. females (2) 0.015 0.025 comparing the autoantibody-positive children and their B. ovatus females (2) 0.31 0.178 corresponding control subjects in the TRIGR cohort (P = *Butyrate-producing bacteria. 0.028 and 0.034, respectively; Mann-Whitney U test). diabetes.diabetesjournals.org DIABETES, VOL. 62, APRIL 2013 1241 FECAL MICROBIOTA COMPOSITION DISCUSSION autoimmunity in the progression of the autoimmune pro- cess toward b-cell destruction and clinical disease. It We observed significant differences in the composition of should be emphasized that our findings demonstrate only fecal microbiota between children positive for at least microbial changes and functional studies are needed to two diabetes-associated autoantibodies and autoantibody- prove causality between these kinds of changes and b-cell negative children. Because the major histocompatability autoimmunity. The results thus are tentative and support complex genotype may affect the intestinal microbiota the animal studies in which causality has been demon- composition (29), we matched the case and control sub- strated. jects not only for age, sex, and early feeding history but There is increasing evidence that the Bifidobacterium also for their HLA class II risk genotype of T1D. Our results genus in the human gut plays an important role in main- thus should not be secondary to HLA-related differences in taining health, both within the gastrointestinal tract and in gut microbiota. the rest of the body (38), and this also is supported by the We found that the score reflecting the abundance of current observations, although we did not provide any data several lactate- and butyrate-producing bacteria, i.e., PC1, on functional changes related to the observed bacterial di- was inversely related to the number of b-cell autoanti- versity. It has been shown that besides contributing to the bodies in children; the lowest levels were observed in production of butyrate, bifidobacteria inhibit bacterial children positive for three or four autoantibodies. The translocation (39–41). In contrast, bacterial translocation bacteria, which correlated with PC1 included B. ado- was found to be enhanced by the Bacteroides genus and, lescentis, an acetate- and lactate-producing bacterium, in particular, the Bacteroides fragilis group, which in- R. faecis, a member from Clostridium cluster XIVa, which cludes B. fragilis, B. ovatus,and B. vulgatus (40). A role produces butyrate using acetate (30), and F. prausnitzii,a of the Bacteroides genus in the development of autoim- member from Clostridium cluster IV, an acetate utilizing mune diabetes has been implicated in animal models and butyrate-producing bacterium (30,31) with anti-inflammatory in humans (11,12,14,33). In our study, the Bacteroides ge- properties (32). In addition to this, we found that in children nus and C. perfringens, which is known to be associated from the FINDIA study, E. hallii, an acetate- and lactate- with increased gut permeability and inflammation via its utilizing and butyrate-producing bacterium from Clostrid- production of several toxins (42), were positively associ- ium cluster XIVa (31), was inversely related to the number ated with b-cell autoimmunity. Bifidobacteria, however, of b-cell autoantibodies. A recent study that included four might inhibit the translocation or the growth of the pairs of cases with development of T1D and autoantibody- B. fragilis group and C. perfringens as they compete for negative control subjects suggested that higher propor- space/adherence (43) and nutrients (44), and they enhance tions of butyrate-producing and mucin-degrading bacteria the intestinal epithelial barrier function (45) by increasing were observed in control subjects compared with case the thickness of the mucus layer (46,47). Accordingly, our subjects (33). Because lactate can be further metabolized findings actually may be interrelated because a low to butyrate, the producers of lactate may contribute to the abundance of bifidobacteria might favor the growth of net production of butyrate; bifidobacteria represents Bacteroides (40). a prime example of this (31). PCA analysis revealed that a Concerning the biological importance of the Bacteroides low abundance (,12%) of the sum of the two most com- genus, it should be noted that its abundance as reported mon bifidobacteria, B. adolescentis and B. pseudocatenu- here obtained via pyrosequencing (;3%) greatly under- latum, was associated with b-cell autoimmunity. estimates its actual predominance within the gut of these Butyrate is thought to be beneficial because it is the children. The reason for this is that the primer pair used in main energy source for colonic epithelial cells (34). Fur- this study, which was chosen because it provides a more thermore, butyrate has been shown to regulate the as- complete assessment of actinobacterial abundance (20), sembly of tight junctions and gut permeability (35). In very likely caused an overestimation of the amount of the animal models of autoimmune diabetes, increased gut Bifidobacterium genus and possibly caused an under- permeability precedes the development of diabetes, and estimation of the Bacteroides genus. We confirmed our environmental factors, which modulate the permeability, suspicions using fluorescent in situ hybridization (data modulate the disease incidence. Although butyrate treat- not shown); the Bifidobacterium genus had been over- ment during the weaning period in BB-DP rats did not estimated by a factor of two and the Bacteroides genus prevent autoimmune diabetes, it modulated the gut in- was underestimated by a factor of six. flammatory response (36). Both gut permeability and in- In agreement with a previous study in which the di- flammation have been linked to the development of T1D versity decreased with increasing age in the four children in humans (37). In children with T1D, subclinical small with development of T1D (14), the microbial diversity was intestinal inflammation with T-cell activation has been lower in autoantibody-positive children when compared reported previously (37), but there are no studies of chil- with autoantibody-negative children, especially in the dren at risk for T1D, i.e., autoantibody-positive children. children aged 12 to 14 years from the TRIGR pilot study. We did not find increased fecal calprotectin or IgA levels The samples from the children of the TRIGR and FINDIA in b-cell autoantibody–positive children, which does not studies were collected at the same time using the same support the presence of significant intestinal inflammation protocol for transportation and storage. Thus, the findings in preclinical T1D, although differences in the composition are not influenced by differences in the storage time or of fecal microbiota were observed. transportation history of the samples. Our results suggest that the changes characterized by In conclusion, our study emphasizes the importance of low numbers of butyrate producers are related to the late bifidobacteria and lactate- or butyrate-producing species in phase of prediabetes, i.e., positivity for multiple autoanti- general in relation to the development of b-cell autoimmu- bodies and IA-2A. The correlation of certain bacterial nity. Bifidobacteria not only supply butyrate-producing findings with the number of positive autoantibodies could species with lactate and acetate but also enhance the in- indicate a role of dysbiosis as a regulator of b-cell testinal epithelial barrier function by modulating the gut 1242 DIABETES, VOL. 62, APRIL 2013 diabetes.diabetesjournals.org M.C. DE GOFFAU AND ASSOCIATES 11. Brugman S, Klatter FA, Visser JT, et al. Antibiotic treatment partially mucosa, thereby possibly preventing the translocation of protects against type 1 diabetes in the Bio-Breeding diabetes-prone rat. Is for example Bacteroides, which were, in turn, confirmed to the gut flora involved in the development of type 1 diabetes? Diabetologia be more highly abundant in children with b-cell autoim- 2006;49:2105–2108 munity. Because only children with at least two autoanti- 12. Roesch LF, Lorca GL, Casella G, et al. Culture-independent identification bodies were included in this study, it was not possible to of gut bacteria correlated with the onset of diabetes in a rat model. ISME J demonstrate that dysbiosis has a role in the initiation of 2009;3:536–548 13. Valladares R, Sankar D, Li N, et al. Lactobacillus johnsonii N6.2 mitigates b-cell autoimmunity. The pyrosequencing method does the development of type 1 diabetes in BB-DP rats. PLoS ONE 2010;5:e10507 have its limitations because it does not give direct data on 14. Giongo A, Gano KA, Crabb DB, et al. Toward defining the autoimmune the functionally important changes of the microbiota, microbiome for type 1 diabetes. ISME J 2011;5:82–91 which should be demonstrated if pathogenetic importance 15. Vaarala O, Ilonen J, Ruohtula T, et al. Removal of bovine insulin from is considered. One also should be careful with making cow’s milk formula and early initiation of beta-cell autoimmunity in the conclusions based on PCA of the data on the bacterial FINDIA Pilot Study. Arch Pediatr Adolesc Med 2012;PMID:22393174 16. Kukko M, Virtanen SM, Toivonen A, et al. Geographical variation in risk diversity. Our results only indicate that alterations in the HLA-DQB1 genotypes for type 1 diabetes and signs of beta-cell autoim- intestinal bacterial diversity are seen at the prediabetes munity in a high-incidence country. Diabetes Care 2004;27:676–681 stage of the disease, and these changes precede the de- 17. Laaksonen M, Pastinen T, Sjöroos M, et al. HLA class II associated risk and velopment of T1D. Large birth cohort studies that include protection against multiple sclerosis-a Finnish family study. J Neuro- time series of samples and methodology to study the immunol 2002;122:140–145 functional changes in the intestinal microbiota are needed 18. Sjöroos M, Iitiä A, Ilonen J, Reijonen H, Lövgren T. Triple-label hybrid- ization assay for type-1 diabetes-related HLA alleles. Biotechniques 1995; for the evaluation of the role of gut microbiota in the de- 18:870–877 velopment of b-cell autoimmunity and T1D. 19. Yu Z, Morrison M. Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques 2004;36:808–812 20. van den Bogert B, de Vos WM, Zoetendal EG, Kleerebezem M. Microarray ACKNOWLEDGMENTS analysis and barcoded pyrosequencing provide consistent microbial pro- Supported by Sigrid Juselius Foundation and the Päivikki files depending on the source of human intestinal samples. Appl Environ Microbiol 2011;77:2071–2080 and Sakari Sohlberg Foundation. 21. Wu GD, Lewis JD, Hoffmann C, et al. Sampling and pyrosequencing No potential conflicts of interest relevant to this article methods for characterizing bacterial communities in the human gut using were reported. 16S sequence tags. BMC Microbiol 2010;10:206 M.C.d.G. and K.L. analyzed the data. M.C.d.G., K.L., and 22. De Filippo C, Cavalieri D, Di Paola M, et al. Impact of diet in shaping gut O.V. wrote the manuscript. M.C.d.G., G.W.W., and H.J.H. microbiota revealed by a comparative study in children from Europe and were responsible for the microbiological studies. K.L. and rural Africa. Proc Natl Acad Sci USA 2010;107:14691–14696 23. Goecks J, Nekrutenko A, Taylor J; Galaxy Team. Galaxy: a comprehensive T.R. coordinated the study recruitment and sample collec- approach for supporting accessible, reproducible, and transparent com- tion. M.K. contributed to the study recruitment and edited putational research in the life sciences. Genome Biol 2010;11:R86 the manuscript. M.K. and T.H. were responsible for the 24. Cole JR, Wang Q, Cardenas E, et al. The Ribosomal Database Project: autoantibody analyses. J.I. was responsible for HLA typing. improved alignments and new tools for rRNA analysis. Nucleic Acids Res L.O. performed the fecal IgA determinations. S.H. per- 2009;37(Database issue):D141–D145 formed the fecal calprotectin tests. O.V. was responsible 25. Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. for the study design. O.V. is the guarantor of this work and, Appl Environ Microbiol 2007;73:5261–5267 as such, had full access to all of the data in the study and 26. Ludwig W, Strunk O, Westram R, et al. ARB: a software environment for takes responsibility for the integrity of the data and the sequence data. Nucleic Acids Res 2004;32:1363–1371 accuracy of the data analysis. 27. Pruesse E, Quast C, Knittel K, et al. SILVA: a comprehensive online re- source for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 2007;35:7188–7196 REFERENCES 28. Lehtonen OP, Gråhn EM, Ståhlberg TH, Laitinen LA. Amount and avidity of 1. Eisenbarth GS. Type I diabetes mellitus. A chronic autoimmune disease. N salivary and serum antibodies against Streptococcus mutans in two groups Engl J Med 1986;314:1360–1368 of human subjects with different dental caries susceptibility. Infect Immun 2. Knip M, Siljander H. Autoimmune mechanisms in type 1 diabetes. Auto- 1984;43:308–313 immun Rev 2008;7:550–557 29. Toivanen P, Vaahtovuo J, Eerola E. Influence of major histocompatibility 3. Knip M, Virtanen SM, Seppä K, et al; Finnish TRIGR Study Group. Dietary complex on bacterial composition of fecal flora. Infect Immun 2001;69: intervention in infancy and later signs of beta-cell autoimmunity. N Engl J 2372–2377 Med 2010;363:1900–1908 30. Duncan SH, Barcenilla A, Stewart CS, Pryde SE, Flint HJ. Acetate utili- 4. Norris JM, Barriga K, Klingensmith G, et al. Timing of initial cereal ex- zation and butyryl coenzyme A (CoA):acetate-CoA transferase in butyrate- posure in infancy and risk of islet autoimmunity. JAMA 2003;290:1713– producing bacteria from the human large intestine. Appl Environ Microbiol 1720 2002;68:5186–5190 5. Vaarala O, Knip M, Paronen J, et al. Cow’s milk formula feeding induces 31. Duncan SH, Louis P, Flint HJ. Lactate-utilizing bacteria, isolated from primary immunization to insulin in infants at genetic risk for type 1 di- human feces, that produce butyrate as a major fermentation product. Appl abetes. Diabetes 1999;48:1389–1394 Environ Microbiol 2004;70:5810–5817 6. Turley SJ, Lee JW, Dutton-Swain N, Mathis D, Benoist C. Endocrine self 32. Sokol H, Pigneur B, Watterlot L, et al. Faecalibacterium prausnitzii is and gut non-self intersect in the pancreatic lymph nodes. 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Does the intestinal 2000;47:646–652 bifidobacterial colonisation affect bacterial translocation? Anaerobe 2008; 46. Kleessen B, Hartmann L, Blaut M. Fructans in the diet cause alterations of 14:43–48 intestinal mucosal architecture, released mucins and mucosa-associated 41. Wang ZT, Yao YM, Xiao GX, Sheng ZY. Risk factors of development of bifidobacteria in gnotobiotic rats. Br J Nutr 2003;89:597–606 gut-derived bacterial translocation in thermally injured rats. World J 47. Kleessen B, Blaut M. Modulation of gut mucosal biofilms. Br J Nutr 2005;93 Gastroenterol 2004;10:1619–1624 (Suppl 1):S35–S40 1244 DIABETES, VOL. 62, APRIL 2013 diabetes.diabetesjournals.org http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diabetes Pubmed Central

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

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Pubmed Central
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© 2013 by the American Diabetes Association.
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0012-1797
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1939-327X
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10.2337/db12-0526
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Abstract

ORIGINAL ARTICLE Fecal Microbiota Composition Differs Between Children With b-Cell Autoimmunity and Those Without 1 2 3,4,5 6,7 2 Marcus C. de Goffau, Kristiina Luopajärvi, Mikael Knip, Jorma Ilonen, Terhi Ruohtula, 3 2 2 1 1 Taina Härkönen, Laura Orivuori, Saara Hakala, Gjalt W. Welling, Hermie J. Harmsen, and Outi Vaarala role of the gut immune system in the pathogenesis of T1D The role of the intestinal microbiota as a regulator of autoim- has been supported by studies showing an immunological mune diabetes in animal models is well-established, but data on link between the pancreas and the gastrointestinal tract. It human type 1 diabetes are tentative and based on studies including only a few study subjects. To exclude secondary effects has been demonstrated that oral antigens are capable of of diabetes and HLA risk genotype on gut microbiota, we activating antigen-specific T cells in pancreatic lymph compared the intestinal microbiota composition in children with nodes (6) and that the interaction between endothelium at least two diabetes-associated autoantibodies (n = 18) with and T cells is controlled by shared homing receptors in autoantibody-negative children matched for age, sex, early feed- inflamed islets and in the gut (7). The development of ing history, and HLA risk genotype using pyrosequencing. Princi- autoimmune diabetes in animal models is regulated by pal component analysis indicated that a low abundance of factors affecting the function of the gut immune system, lactate-producing and butyrate-producing species was associated such as dietary factors and microbial stimuli, which fur- with b-cell autoimmunity. In addition, a dearth of the two most ther affect the intestinal mucosal barrier and immune re- dominant Bifidobacterium species, Bifidobacterium adolescentis and Bifidobacterium pseudocatenulatum, and an increased sponsiveness (8). The effects of intestinal microbes may abundance of the Bacteroides genus were observed in the chil- not be restricted to barrier mechanisms, but gut micro- dren with b-cell autoimmunity. We did not find increased fecal biota seems to play a key role in the regulation of the T-cell calprotectin or IgA as marker of inflammation in children with populations in the gut, including regulatory T cells, T b-cell autoimmunity. Functional studies related to the observed helper 1, and T helper 17 cells (9). alterations in the gut microbiome are warranted because the low Several animal studies indicate that alterations in the abundance of bifidobacteria and butyrate-producing species intestinal microbiota are associated with the development could adversely affect the intestinal epithelial barrier function of autoimmune diabetes. Nonobese diabetic mice lacking and inflammation, whereas the apparent importance of the Bac- MyD88, an essential signal transducer in Toll-like receptor teroides genus in development of type 1 diabetes is insufficiently signaling, did not have development of diabetes (10), understood. Diabetes 62:1238–1244, 2013 which emphasizes the role of intestinal microbiota as a regulator of autoimmune diabetes. There are differences in the gut microbiota between bio-breeding (BB) diabetes- ype 1 diabetes (T1D) is caused by the destruction prone (DP), and diabetes-resistant rats before the diagnosis of the pancreatic b-cells in genetically suscepti- of diabetes. Antibiotics also can prevent autoimmune di- ble individuals. The disease is considered to be abetes in BB-DP rats (11). Furthermore, it has been Timmune mediated, and the appearance of circu- reported that stool from BB diabetes-resistant rats con- lating autoantibodies against b-cells is seen years before tained more probiotic-like bacteria, whereas Bacteroides, the diagnosis along with a significant reduction in b-cell Eubacterium, and Ruminococcus were more prevalent in mass (1,2). Environmental factors associated with the ac- BB-DP rats (12). Lactobacillus johnsonii prevented di- tivation of the gut immune system, such as early exposure abetes when administered to BB-DP rats (13). There are to dietary antigens (cow’s milk and gluten), have been only a few studies of the intestinal microbiota in relation to associated with the induction of this process (3–5). The T1D in humans, but the results of a follow-up study in- cluding four children with development of T1D suggested that the Bacteroidetes-to-Firmicutes ratio increased over From the Department of Medical Microbiology, University Medical Center time in those children with eventual progression to clinical Groningen and University of Groningen, Groningen, the Netherlands; the T1D, whereas it decreased in children who remained Immune Response Unit, Department of Vaccination and Immune Protec- nondiabetic (14). tion, National Institute for Health and Welfare, Helsinki, Finland; the Children’s Hospital, University of Helsinki and Helsinki University Central The aim of this study was to compare the composition of Hospital, Helsinki, Finland; the Folkhälsan Research Center, Helsinki, Fin- the gut microbiota between children with b-cell autoim- land; the Department of Pediatrics, Tampere University Hospital, Tampere, munity and autoantibody-negative children matched for Finland; the Immunogenetics Laboratory, University of Turku, Turku, Finland; and the Department of Clinical Immunology, University of Eastern age, sex, HLA risk genotype, and early feeding history Finland, Kuopio, Finland. using pyrosequencing as the method of choice. Corresponding author: Outi Vaarala, outi.vaarala@thl.fi. Received 2 May 2012 and accepted 2 November 2012. DOI: 10.2337/db12-0526. Clinical trial reg. nos. NCT00570102 and NCT01055080, clinicaltrials.gov. RESEARCH DESIGN AND METHODS This article contains Supplementary Data online at http://diabetes The current study included 18 children with HLA-conferred susceptibility to .diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0526/-/DC1. T1D who had development of signs of progressive b-cell autoimmunity, i.e., M.C.d.G. and K.L. contributed equally to this study. tested positive for at least two diabetes-associated autoantibodies (cases). 2013 by the American Diabetes Association. Readers may use this article as Eighteen control children were matched for age, sex, and HLA-DQB1 geno- long as the work is properly cited, the use is educational and not for profit, type, as well as for the time of exposure to and the type of infant formula. The and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. characteristics of the children recruited to the gut microbiota study are shown 1238 DIABETES, VOL. 62, APRIL 2013 diabetes.diabetesjournals.org M.C. DE GOFFAU AND ASSOCIATES in Table 1 and are shown in detail in Supplementary Table 1. The study sub- positive for three autoantibodies, and five were positive for two autoanti- jects were recruited from the study population of two intervention trials bodies (Table 1). performed in Finland: the Trial to Reduce IDDM in the Genetically at Risk HLA genotyping. HLA genotyping was performed according to the screening (TRIGR) pilot (n = 20) or the Finnish Dietary Intervention Trial for Prevention protocol in the TRIGR and FINDIA studies. The initial HLA-DQB1 typing for of Type 1 Diabetes (FINDIA) pilot study (n = 16) (3,15). In the TRIGR pilot risk-associated (DQB1*02, DQB1*03:02) and protective (DQB1*03:01, study, autoantibody positivity was monitored until the age of 10 years and in DQB1*06:02, and DQB1*06:03) alleles was complemented with DQA1 typing the ongoing FINDIA pilot study and follow-up time for autoantibodies varied for DQA1*02:01 and DQA1*05 alleles in those with DQB1*02 without pro- from 3 to 6 years. Eighteen of 26 children who had development of at least two tective alleles or the major risk allele DQB1*03:02. This two-step screening autoantibodies in these intervention studies but who had not yet had pro- technique is based on the hybridization of PCR products with lanthanide- gression to overt T1D participated in the current study. The fecal samples labeled probes detected by time-resolved fluorometry as described previously from the study subjects recruited from the FINDIA and TRIGR studies were (17,18). similarly collected between March 2009 and February 2010. Fecal samples DNA extraction. Total DNA was extracted from 0.25 g fecal sample using the from children were collected using stool collection vials and immediately repeated bead beating method described in detail by Yu et al. (19), with stored in home freezers (220°C). Families delivered the frozen sample to the a number of modifications. In brief, four 3-mm glass beads were added during study center, and the sample was stored at 280°C until processing. The fecal the homogenization step, whereas 0.5-mm glass beads were not used at all. samples were collected at a point in time when the study subjects did not have Bead beating was performed using a Precellys 24 (Bertin Technologies, gastroenteritis and had not received any antibiotic treatment during the past 3 Montigny le Bretonneux, France) at 5.5 ms in three rounds of 1 min each months. Four children had developed T1D after the fecal samples were col- with 30-s pauses at room temperature in between. The incubation temperature lected. The control children remained negative for T1D and for all four after the bead beating was increased from 70°C to 95°C. Importantly, protein autoantibodies analyzed. The study was approved by the ethics committees of precipitation with 260 mL ammonium acetate was performed twice. Elution of the participating hospitals and the families gave their written informed con- DNA from the purification columns was performed twice. Columns from the sent. QiaAmp Stool kit were replaced by those from the QIAamp DNA Mini Kit In the TRIGR pilot study, infants with a first-degree relative affected by T1D (Qiagen, Hilden, Germany). were randomized to receive either a regular cow’s milk formula (Enfamil; Pyrosequencing. From each sample, the 16S rRNA genes were amplified using Mead Johnson, Evansville, IN) or an extensively hydrolyzed casein-based test a primer set corresponding to primers 27F-degS (20) and 534-R (21). These formula (Nutramigen; Mead Johnson) until the age of 6–8 months. In the PCR primers target the V1, V2, and V3 hypervariable regions of the 16 S rRNA; FINDIA study, infants were randomized to receive a standard cow’s milk– 27-degS was chosen in particular because it appears to provide a more com- based formula (Tutteli; Valio, Helsinki, Finland), a whey-based hydrolyzed plete assessment of actinobacterial abundance (20). Pyrosequencing was formula (Peptidi-Tutteli), or a whey-based FINDIA formula from which bo- performed using a Roche FLX Genome Sequencer at DNAvision (Liège, Bel- vine insulin was removed. In both studies exclusive breastfeeding was en- gium) using their standard protocol (22). couraged. Sequencing quality control. Pyrosequencing produced a total of 461,874 Autoantibody assays. Insulin autoantibodies, autoantibodies against the 65- reads of 16S rDNA. Sequences were assigned to samples according to sample- kDa isoform of glutamic acid decarboxylase, and autoantibodies against the specific barcodes. Using the Galaxy Tools web site (23), SFF files from the 454 protein tyrosinase phosphatase–related IA-2 molecule (IA-2A) were mea- Genome Sequencer FLX were converted into FASTA files and FASTA quality sured by specific radiobinding assays, and islet cell antibodies were mea- files. FASTA formatted files contained an average (6 SD) of 12,830 6 4,888 sured by a standard immunofluorescence assay as described previously (16). reads per sample. The RDP pyrosequencing pipeline (24) (RDP 10 database, Six out of 18 cases tested positive for four autoantibodies, seven were update 17) was subsequently used to check the FASTA sequence files for the same criteria as described by De Filippo et al. (22) and to check that the av- erage experimental quality score was at least 20. After this quality check, the FASTA files contained an average (6 SD) of 8,024 6 3,136 high-quality reads. TABLE 1 Classification. Taxonomy (phylum, family, and genus level) was assigned Characteristics of the study subjects using RDP classifier 2.01 (25). Richness and diversity analyses were performed as described by De Filippo et al. (22). Identification to the species level was Case children Control children performed using ARB software (26). For this, SSU reference database Characteristics (n) (n = 18) (n = 18) (SSURef_106_ SILVA_19_03_11) was downloaded from the SILVA web site (27). From this database, only sequences of cultured and identified isolates Female/male 7/11 7/11 were used. From these sequences, a “PT-server” database was built, which T1D in first-degree was subsequently used to find the closest match for each of our high-quality relative 10 10 sequences imported from the FASTA files. For this, the “search next relatives Age (years) of listed species in PT-server” function was applied with the following settings: TRIGR pilot study 13.3 (11.7214.2) 12.8 (11.9213.6) oligo length, 12; mismatches, 0; match score, relative; and minimum score, 10. FINDIA pilot study 5.1 (4.926.0) 5.0 (3.9–7.0) The average (6 SD) match score was 75.2 6 18.5. Sequences that were HLA-DQB1 genotype identified as being from different strains but belonging to the same species were grouped together. Species that represented .0.005% of all sequences *02/03:02 7 7 were taken along for statistical analysis, together representing 99.2 6 0.35% of *03:02/x 8 8 all high-quality FASTA reads per sample. *02(DQA1*05)/y 2 2 Assay of IgA and calprotectin in stool samples. A beaker in the bottom cap *02(DQA1*02:01) 1 1 of the extraction device was filled with thawed and homogenized stool sample, Study formula avoiding seeds and grains (;100 mg). The extraction tube was filled with 4.9 CM 10 10 mL extraction buffer and vortexed for 30 s. Mixing was continued in a shaker HC 4 4 at 1,000 rpm for 3 min. The particles were allowed to settle before 10-min HW 3 3 centrifugation at 10,000g at room temperature. Supernatant was collected and FINDIA 1 1 stored at 220°C. Duration of exclusive IgA values were measured with the modified ELISA method described by BF (mo) 2.9 (025.5) 4.0 (0.126.0) Lehtonen et al. (28). Calprotectin levels in stool samples were determined using Calprolab calprotectin ELISA tests (Lot CALP-Pilot3; Calpro AS, Total duration of BF Lysaker, Norway); 20 mL extract was mixed with 980 mL of sample dilution (mo) 8.1 (1.6–16.5) 10.5 (5.1–19.3), P = 0.03 buffer. Cesarean delivery 4 3 Statistical analysis. Principal component analysis (PCA) was performed to find clusters of similar groups of samples or species. PCA is an ordination Cases are children positive for at least two diabetes-associated auto- method based on multivariate statistical analysis that maps the samples in antibodies and control subjects are negative for b-cell autoantibod- ies. The study subjects were participants in the TRIGR and FINDIA different dimensions. All tests were performed with PASW Statistics 18 (SPSS, pilot studies. Data are number or medians (with range). x  *03:01 or Chicago, IL). Initial analysis of the samples showed that bacterial populations *06:02, y  *03:01, *06:02, or *06:03. BF, breastfeeding; CM, cow’s were most often not normally distributed. Mann-Whitney U and Spearman r milk formula; FINDIA, insulin-free whey-based formula; HC, hydro- and x tests were used. All tests were two-tailed. P , 0.05 was considered to lyzed casein-based formula; HW, hydrolyzed whey-based formula. indicate statistical significance. diabetes.diabetesjournals.org DIABETES, VOL. 62, APRIL 2013 1239 FECAL MICROBIOTA COMPOSITION RESULTS important observation illustrated in Fig. 2. PC2 showed an inverse correlation with the abundance of B. adolescentis, Comparison of sequence diversity at phylum, family, but a positive correlation with the abundance of Bifido- and genus levels. The analysis of the high-quality reads of bacterium pseudocatenulatum (P =1 3 10 and P =1 3 all samples at the phylum level showed that Firmicutes 10 ; Spearman r test). B. adolescentis and B. pseudoca- (58.1%), Actinobacteria (36.2%), and Bacteroidetes (3.4%) tenulatum represented the two most commonly identified were the most dominant phyla. On the family level, the species (11.0 and 9.1%, respectively). The PC2 score dif- Bifidobacteriaceae (32.8%; Actinobacteria), the Lachnospir- fered significantly between the children from the TRIGR aceae (18.4%; Firmicutes), and Ruminococcaceae (17.1%; study representing older children and the FINDIA children Firmicutes) were the most common. On the genus level, who were younger (P = 0.001; Mann Whitney U test). the Bifidobacterium genus was the most frequent B. adolescentis was the most common species (15.8%) (34.2%). The most interesting finding was that the Bac- among the children in the TRIGR pilot study, and their teroidetes phylum, the Bacteroidaceae family (2.5%), and samples were clustered in the left leg of the chevron-like the Bacteroides genus (3.1%) were more common in autoantibody-positive children than in autoantibody- distribution, whereas B. pseudocatenulatum was most frequent (15.8%) in the children from the FINDIA study, negative peers (4.6 vs. 2.2%, 3.5 vs. 1.5%, and 4.3 vs. 2.0%, respectively; P = 0.035, 0.022, and 0.031, respectively; and their samples were clustered in the right leg. PC3 was inversely related to the abundance of both B. adolescentis Mann-Whitney U test). Species level PCA. PCA analysis of the species level and B. pseudocatenulatum (P = 0.023 and P = 0.002; Spearman r test), and it is characterized by the sum of revealed various correlations with b-cell autoimmunity. The first principal component (PC1) (46.0%) correlated B. adolescentis and B. pseudocatenulatum (P =1 3 10 ; Spearman r test). Various bacteria were inversely associ- positively with a number of important short-chain fatty acid–producing species (P # 0.01 for all species; Spear- ated with the combined count of these two bifidobacteria, especially the members of the Clostridium cluster XI (P = man r test), such as Bifidobacterium adolescentis (11%), Faecalibacterium prausnitzii (5.6%), Clostridium clos- 6 3 10 ).The apex, encompassed by a circle in Fig. 2, can be described as comprising those samples in which the tridioforme (2.3%), and Roseburia faecis (0.94%), as shown in Fig. 1. This PC1 was inversely related to the combined abundance of B. adolescentis and B. pseudo- catenulatum is ,12%. The children with b-cell autoim- number of diabetes-associated autoantibodies in children (Fig. 1; P = 0.018; Spearman r test). The children with four munity were over-represented in the apex when compared with control children (10/18 vs. 4/18; P = 0.040; x test). autoantibodies had a significantly lower PC1 score, i.e., they had lower numbers of short-chain fatty acid pro- Species-level analysis. The association of single bacte- rial species with autoantibody positivity is shown in ducers than the control children (P = 0.008; Mann-Whitney U test). PC1 score was especially lower in IA-2A–positive Table 2. Roseburia faecis (0.94%) was more abundant in autoantibody-negative than autoantibody-positive children children than in children negative for IA-2A (P = 0.004; Mann-Whitney U test). (P = 0.009; Mann-Whitney U test), whereas Clostridium per- A remarkable chevron-like distribution of dots in the fringens (0.03%) were more abundant in children with b-cell second PC (PC2) and third PC (PC3) provided another autoimmunity than in those without (P = 0.18; Mann-Whitney FIG. 1. The association of PC1 with autoantibody positivity was demonstrated as an inverse correlation between PC1 and number of b-cell autoantibodies in the study cohort (P = 0.018; Spearman r test) and as a difference in PC1 score between the children positive for four auto- antibodies and control children negative for autoantibodies (P = 0.008; Mann-Whitney test). Control children are indicated by being positive for no autoantibodies and cases are positive for two, three, or four autoantibodies (x-axis). Correlations between PC1 and bacterial groups are shown in right panel. 1240 DIABETES, VOL. 62, APRIL 2013 diabetes.diabetesjournals.org M.C. DE GOFFAU AND ASSOCIATES FIG. 2. The distribution of children positive for b-cell autoantibodies (case subjects with open symbols) and autoantibody-negative children (control subjects with filled symbols) according to PC2 (x-axis) and PC3 (y-axis). PC2 shows an inverse correlation with the abundance of B. adolescentis and a positive correlation with B. pseudocatenulatum, whereas PC3 shows an inverse correlation with both B. adolescentis and B. pseudocatenulatum. Indicated percentages indicate the prevalence of B. adolescentis and B. pseudocatenulatum, respectively. Age of the chil- dren is associated with PC2, whereas autoantibody positivity is associated with PC3. The children 3 to 7 years of age (circle) from the FINDIA pilot have higher numbers of B. pseudocatenulatum and lower numbers of B. adolescentis than the children from the TRIGR pilot 11 to 14 years of age (square). The main correlations with PC2 and PC3 are depicted with vectors in the top left. Children with a dearth of both B. adolescentis and B. pseudocatenulatum (sum <12%) are encompassed by the dashed circle near the top and the number of autoantibody-positive children is higher than the number of control subjects at the apex (10/18 vs. 4/18; P = 0.040; x test). U test). The Bacteroides genus was associated with auto- In children from the FINDIA study, it is furthermore antibody positivity, but only a few of its many members noteworthy that Eubacterium hallii (6.0%) was strongly were observed to be related on the species level. In- inversely related to the number of b-cell autoantibodies terestingly, the correlation of the Bacteroides genus with (P = 0.002; Spearman r correlation test). autoantibody positivity was significant only in males, not IgA and calprotectin levels. Calprotectin levels, a in females (P = 0.005 vs. 0.655, respectively). marker of intestinal inflammation, were not observed to be correlated with autoantibody positivity. Clostridium orbiscindens (0.47%) correlated inversely with calpro- TABLE 2 tectin levels (P = 0.004; Spearman r test). Lower levels of Association between single bacterial species and signs of b-cell calprotectin were seen in children with the lower-risk HLA autoimmunity genotype, i.e., (DR3)DQB1*02-DQA1*05, when compared with the children with moderate-risk or high-risk HLA risk Mann-Whitney Variable % U test genotypes (P = 0.001 and 0.005; Mann-Whitney U test). Fecal IgA levels did not differ between autoantibody- *Roseburia faecis (2) 0.94 0.009 positive and autoantibody-negative children but were in- *Clostridium xylanovorans (2) 0.03 0.031 versely correlated with age (P = 0.007; Mann-Whitney U *Peptoniphilus gorbachii (2) 0.006 0.008 test). *Eubacterium hallii (2) 6.0 0.088 Diversity analyses. The analysis of the 90, 95, and 97% *Eubacterium desmolans (2) 0.15 0.058 operational taxonomic similarity levels showed that the *Acetanaerobacterium elongatum (2) 0.06 0.043 diversity was significantly higher in the children in the Bifidobacterium animalis (+) 0.18 0.028 Lactobacillus acidophilus (+) 0.03 0.037 TRIGR pilot cohort than in the children in the FINDIA pilot Clostridium perfringens (+) 0.03 0.018 cohort (P , 0.001; Mann-Whitney U test). Furthermore, Bacteroides genus (+) 3.1 0.031 there was a trend that the diversity per sample was higher Bacteroides genus males (+) 3.6 0.005 in autoantibody-negative children than in the autoantibody- Bacteroides genus females (+) 2.3 0.655 positive children (Supplementary Table 2). This difference B. vulgatus males (+) 1.3 0.023 in diversity, measured in the number of observed opera- B. ovatus males (+) 0.21 0.049 tional taxonomic units and the Chao index, was signifi- B. fragilis females (+) 0.16 0.017 cantly different at the 95% taxonomic similarity level when B. thetaiota. females (2) 0.015 0.025 comparing the autoantibody-positive children and their B. ovatus females (2) 0.31 0.178 corresponding control subjects in the TRIGR cohort (P = *Butyrate-producing bacteria. 0.028 and 0.034, respectively; Mann-Whitney U test). diabetes.diabetesjournals.org DIABETES, VOL. 62, APRIL 2013 1241 FECAL MICROBIOTA COMPOSITION DISCUSSION autoimmunity in the progression of the autoimmune pro- cess toward b-cell destruction and clinical disease. It We observed significant differences in the composition of should be emphasized that our findings demonstrate only fecal microbiota between children positive for at least microbial changes and functional studies are needed to two diabetes-associated autoantibodies and autoantibody- prove causality between these kinds of changes and b-cell negative children. Because the major histocompatability autoimmunity. The results thus are tentative and support complex genotype may affect the intestinal microbiota the animal studies in which causality has been demon- composition (29), we matched the case and control sub- strated. jects not only for age, sex, and early feeding history but There is increasing evidence that the Bifidobacterium also for their HLA class II risk genotype of T1D. Our results genus in the human gut plays an important role in main- thus should not be secondary to HLA-related differences in taining health, both within the gastrointestinal tract and in gut microbiota. the rest of the body (38), and this also is supported by the We found that the score reflecting the abundance of current observations, although we did not provide any data several lactate- and butyrate-producing bacteria, i.e., PC1, on functional changes related to the observed bacterial di- was inversely related to the number of b-cell autoanti- versity. It has been shown that besides contributing to the bodies in children; the lowest levels were observed in production of butyrate, bifidobacteria inhibit bacterial children positive for three or four autoantibodies. The translocation (39–41). In contrast, bacterial translocation bacteria, which correlated with PC1 included B. ado- was found to be enhanced by the Bacteroides genus and, lescentis, an acetate- and lactate-producing bacterium, in particular, the Bacteroides fragilis group, which in- R. faecis, a member from Clostridium cluster XIVa, which cludes B. fragilis, B. ovatus,and B. vulgatus (40). A role produces butyrate using acetate (30), and F. prausnitzii,a of the Bacteroides genus in the development of autoim- member from Clostridium cluster IV, an acetate utilizing mune diabetes has been implicated in animal models and butyrate-producing bacterium (30,31) with anti-inflammatory in humans (11,12,14,33). In our study, the Bacteroides ge- properties (32). In addition to this, we found that in children nus and C. perfringens, which is known to be associated from the FINDIA study, E. hallii, an acetate- and lactate- with increased gut permeability and inflammation via its utilizing and butyrate-producing bacterium from Clostrid- production of several toxins (42), were positively associ- ium cluster XIVa (31), was inversely related to the number ated with b-cell autoimmunity. Bifidobacteria, however, of b-cell autoantibodies. A recent study that included four might inhibit the translocation or the growth of the pairs of cases with development of T1D and autoantibody- B. fragilis group and C. perfringens as they compete for negative control subjects suggested that higher propor- space/adherence (43) and nutrients (44), and they enhance tions of butyrate-producing and mucin-degrading bacteria the intestinal epithelial barrier function (45) by increasing were observed in control subjects compared with case the thickness of the mucus layer (46,47). Accordingly, our subjects (33). Because lactate can be further metabolized findings actually may be interrelated because a low to butyrate, the producers of lactate may contribute to the abundance of bifidobacteria might favor the growth of net production of butyrate; bifidobacteria represents Bacteroides (40). a prime example of this (31). PCA analysis revealed that a Concerning the biological importance of the Bacteroides low abundance (,12%) of the sum of the two most com- genus, it should be noted that its abundance as reported mon bifidobacteria, B. adolescentis and B. pseudocatenu- here obtained via pyrosequencing (;3%) greatly under- latum, was associated with b-cell autoimmunity. estimates its actual predominance within the gut of these Butyrate is thought to be beneficial because it is the children. The reason for this is that the primer pair used in main energy source for colonic epithelial cells (34). Fur- this study, which was chosen because it provides a more thermore, butyrate has been shown to regulate the as- complete assessment of actinobacterial abundance (20), sembly of tight junctions and gut permeability (35). In very likely caused an overestimation of the amount of the animal models of autoimmune diabetes, increased gut Bifidobacterium genus and possibly caused an under- permeability precedes the development of diabetes, and estimation of the Bacteroides genus. We confirmed our environmental factors, which modulate the permeability, suspicions using fluorescent in situ hybridization (data modulate the disease incidence. Although butyrate treat- not shown); the Bifidobacterium genus had been over- ment during the weaning period in BB-DP rats did not estimated by a factor of two and the Bacteroides genus prevent autoimmune diabetes, it modulated the gut in- was underestimated by a factor of six. flammatory response (36). Both gut permeability and in- In agreement with a previous study in which the di- flammation have been linked to the development of T1D versity decreased with increasing age in the four children in humans (37). In children with T1D, subclinical small with development of T1D (14), the microbial diversity was intestinal inflammation with T-cell activation has been lower in autoantibody-positive children when compared reported previously (37), but there are no studies of chil- with autoantibody-negative children, especially in the dren at risk for T1D, i.e., autoantibody-positive children. children aged 12 to 14 years from the TRIGR pilot study. We did not find increased fecal calprotectin or IgA levels The samples from the children of the TRIGR and FINDIA in b-cell autoantibody–positive children, which does not studies were collected at the same time using the same support the presence of significant intestinal inflammation protocol for transportation and storage. Thus, the findings in preclinical T1D, although differences in the composition are not influenced by differences in the storage time or of fecal microbiota were observed. transportation history of the samples. Our results suggest that the changes characterized by In conclusion, our study emphasizes the importance of low numbers of butyrate producers are related to the late bifidobacteria and lactate- or butyrate-producing species in phase of prediabetes, i.e., positivity for multiple autoanti- general in relation to the development of b-cell autoimmu- bodies and IA-2A. The correlation of certain bacterial nity. Bifidobacteria not only supply butyrate-producing findings with the number of positive autoantibodies could species with lactate and acetate but also enhance the in- indicate a role of dysbiosis as a regulator of b-cell testinal epithelial barrier function by modulating the gut 1242 DIABETES, VOL. 62, APRIL 2013 diabetes.diabetesjournals.org M.C. DE GOFFAU AND ASSOCIATES 11. Brugman S, Klatter FA, Visser JT, et al. Antibiotic treatment partially mucosa, thereby possibly preventing the translocation of protects against type 1 diabetes in the Bio-Breeding diabetes-prone rat. Is for example Bacteroides, which were, in turn, confirmed to the gut flora involved in the development of type 1 diabetes? Diabetologia be more highly abundant in children with b-cell autoim- 2006;49:2105–2108 munity. Because only children with at least two autoanti- 12. Roesch LF, Lorca GL, Casella G, et al. Culture-independent identification bodies were included in this study, it was not possible to of gut bacteria correlated with the onset of diabetes in a rat model. ISME J demonstrate that dysbiosis has a role in the initiation of 2009;3:536–548 13. Valladares R, Sankar D, Li N, et al. Lactobacillus johnsonii N6.2 mitigates b-cell autoimmunity. The pyrosequencing method does the development of type 1 diabetes in BB-DP rats. PLoS ONE 2010;5:e10507 have its limitations because it does not give direct data on 14. Giongo A, Gano KA, Crabb DB, et al. Toward defining the autoimmune the functionally important changes of the microbiota, microbiome for type 1 diabetes. ISME J 2011;5:82–91 which should be demonstrated if pathogenetic importance 15. Vaarala O, Ilonen J, Ruohtula T, et al. Removal of bovine insulin from is considered. One also should be careful with making cow’s milk formula and early initiation of beta-cell autoimmunity in the conclusions based on PCA of the data on the bacterial FINDIA Pilot Study. Arch Pediatr Adolesc Med 2012;PMID:22393174 16. Kukko M, Virtanen SM, Toivonen A, et al. Geographical variation in risk diversity. Our results only indicate that alterations in the HLA-DQB1 genotypes for type 1 diabetes and signs of beta-cell autoim- intestinal bacterial diversity are seen at the prediabetes munity in a high-incidence country. Diabetes Care 2004;27:676–681 stage of the disease, and these changes precede the de- 17. Laaksonen M, Pastinen T, Sjöroos M, et al. HLA class II associated risk and velopment of T1D. Large birth cohort studies that include protection against multiple sclerosis-a Finnish family study. J Neuro- time series of samples and methodology to study the immunol 2002;122:140–145 functional changes in the intestinal microbiota are needed 18. Sjöroos M, Iitiä A, Ilonen J, Reijonen H, Lövgren T. Triple-label hybrid- ization assay for type-1 diabetes-related HLA alleles. Biotechniques 1995; for the evaluation of the role of gut microbiota in the de- 18:870–877 velopment of b-cell autoimmunity and T1D. 19. Yu Z, Morrison M. Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques 2004;36:808–812 20. van den Bogert B, de Vos WM, Zoetendal EG, Kleerebezem M. Microarray ACKNOWLEDGMENTS analysis and barcoded pyrosequencing provide consistent microbial pro- Supported by Sigrid Juselius Foundation and the Päivikki files depending on the source of human intestinal samples. Appl Environ Microbiol 2011;77:2071–2080 and Sakari Sohlberg Foundation. 21. Wu GD, Lewis JD, Hoffmann C, et al. Sampling and pyrosequencing No potential conflicts of interest relevant to this article methods for characterizing bacterial communities in the human gut using were reported. 16S sequence tags. BMC Microbiol 2010;10:206 M.C.d.G. and K.L. analyzed the data. M.C.d.G., K.L., and 22. De Filippo C, Cavalieri D, Di Paola M, et al. Impact of diet in shaping gut O.V. wrote the manuscript. M.C.d.G., G.W.W., and H.J.H. microbiota revealed by a comparative study in children from Europe and were responsible for the microbiological studies. K.L. and rural Africa. Proc Natl Acad Sci USA 2010;107:14691–14696 23. Goecks J, Nekrutenko A, Taylor J; Galaxy Team. Galaxy: a comprehensive T.R. coordinated the study recruitment and sample collec- approach for supporting accessible, reproducible, and transparent com- tion. M.K. contributed to the study recruitment and edited putational research in the life sciences. Genome Biol 2010;11:R86 the manuscript. M.K. and T.H. were responsible for the 24. 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Journal

Diabetes – Pubmed Central

Published: Mar 14, 2013

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