3510 Diabetes Volume 64, October 2015 Aimon K. Alkanani,1 Naoko Hara,1 Peter A. Gottlieb,1 Diana Ir,2 Charles E. Robertson,2,3 Brandie D. Wagner,3,4 Daniel N. Frank,2,3 and Danny Zipris1 Alterations in Intestinal Microbiota Correlate With Susceptibility to Type 1 Diabetes Diabetes 2015;64:3510–3520 | DOI: 10.2337/db14-1847 We tested the hypothesis that alterations in the intestinal environmental factors in the disease process. The intesti- microbiota are linked with the progression of type 1 nal microbiota plays a key role in the development and diabetes (T1D). Herein, we present results from a study function of the immune system (2). Data from human performed in subjects with islet autoimmunity living in and animal studies have led to the hypothesis that altered the U.S. High-throughput sequencing of bacterial 16S rRNA gut microbiota (“dysbiosis”) could be associated with genes and adjustment for sex, age, autoantibody pres- mechanisms of metabolic and immune-mediated disor- ence, and HLA indicated that the gut microbiomes of ders, such as obesity, celiac disease, type 2 diabetes, and seropositive subjects differed from those of autoantibody- inflammatory bowel disease (3,4). fi free rst-degree relatives (FDRs) in the abundance of Dysbiosis has been postulated to be associated with four taxa. Furthermore, subjects with autoantibodies, mechanisms of T1D (5–11). The development of T1D in seronegative FDRs, and new-onset patients had differ- animal models, such as the NOD (6) and RIP-B7.1 (12) ent levels of the Firmicutes genera Lactobacillus and mice and the diabetes-prone BioBreeding (13) and Staphylococcus compared with healthy control subjects with no family history of autoimmunity. Further analysis LEW1.WR1 (10) rats, is linked with changes in the intestinal revealed trends toward increased and reduced abundan- microbiome. Human studies performed in subjects at risk for ces of the Bacteroidetes genera Bacteroides and Prevo- T1D provided evidence for a decline and an increase in tella, respectively, in seropositive subjects with multiple the abundance of the bacterial phyla Firmicutes and versus one autoantibody. Canonical discriminant analysis Bacteroidetes, respectively (14). Others reported the absence suggested that the gut microbiomes of autoantibody- of Bifidobacterium species and increased levels of the ge- IMMUNOLOGY AND TRANSPLANTATION positive individuals and seronegative FDRs clustered to- nus Bacteroides in children with two or more islet auto- gether but separate from those of new-onset patients antibodies (15). A study performed in subjects with T1D and unrelated healthy control subjects. Finally, no differ- further demonstrated an increase in the abundances of ences in biodiversity were evident in seropositive versus Bacteroidetes and Clostridium spp. in addition to a reduc- seronegative FDRs. These observations suggest that al- tion in the genera Lactobacillus and Bifidobacterium (16). tered intestinal microbiota may be associated with dis- Finally, significant alterations in microbial interaction ease susceptibility. networks with no differences in bacterial diversity, mic- robial composition, or the level of bacterial genera were observed in seropositive and seronegative children (17). Type 1 diabetes (T1D) is an autoimmune disorder that in- In this study, we tested the hypothesis that alterations volves b-cell inflammation and destruction (1). Although in the human intestinal microbiota are linked with the the mechanisms that trigger the disease are not yet clear, progression of human T1D. Analysis of 16S bacterial rRNA human and animal studies implicate both genetic and sequencing data demonstrated that the abundances of four 1Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Corresponding author: Danny Zipris, [email protected]. Aurora, CO Received 3 December 2014 and accepted 29 May 2015. 2Division of Infectious Diseases, School of Medicine, University of Colorado This article contains Supplementary Data online at http://diabetes Denver, Aurora, CO .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1847/-/DC1. 3University of Colorado Denver Microbiome Research Consortium, School of Medicine, University of Colorado Denver, Aurora, CO © 2015 by the American Diabetes Association. Readers may use this article as 4Department of Biostatistics and Informatics, Colorado School of Public Health, long as the work is properly cited, the use is educational and not for profit, and University of Colorado Denver, Aurora, CO the work is not altered. diabetes.diabetesjournals.org Alkanani and Associates 3511 bacterial genera were altered in seropositive subjects com- 7.9% (range 5.5–15), respectively. Subjects who received pared with seronegative first-degree relatives (FDRs). Further- antibiotic therapy up to 4 weeks prior to sample collection more, seropositive subjects and seronegative FDRs as well as and individuals with known infections or gastrointestinal new-onset patients have a reduction in the abundance of disorders were not included in the study. One subject in each the Firmicutes genera Lactobacillus or Staphylococcus com- of the seropositive and new-onset groups and three from the pared with unrelated healthy control subjects. The data sug- seronegative cohort reported being vegetarians. The study gest that gastrointestinal tract dysbiosis may be associated was approved by the institutional review board at the Uni- with disease progression. versity of Colorado Denver. Fecal samples were collected at the Barbara Davis Center for Diabetes or at the home of the RESEARCH DESIGN AND METHODS study participants using stool collection tubes and stored 2 Subject Characteristics at 80°C until use. Samples collected at home were stored at 220°C and delivered to the Barbara Davis Center for We analyzed the gut microbiome from four subject co- Diabetes on ice by study subjects or laboratory personnel. horts with or without evidence of islet autoimmunity residing in the Denver metro area. These studies included Microbiome Analysis 35 subjects with newly diagnosed T1D (up to 6 months Bacterial profiles were determined by broad-range am- from disease diagnosis) recruited during their routine plification and sequence analysis of 16S rRNA genes. In visits to the Barbara Davis Center for Diabetes. Twenty- brief, amplicons were generated using primers that target one individuals with one to four autoantibodies and 32 ;280 base pairs of the V4 variable region of the 16S seronegative FDRs of subjects with islet autoimmunity rRNA gene (19). PCR products were normalized using a were recruited from the Type 1 Diabetes TrialNet Natural SequalPrep kit (Invitrogen, Carlsbad, CA), pooled, lyoph- History Study (Table 1). Only two seronegative subjects ilized, purified, and concentrated using a DNA Clean & had siblings in the seropositive group. Twenty-three in- Concentrator Kit (Zymo, Irvine, CA). The amplicon pool dividuals without any family history of autoimmunity was quantified using Qubit 2.0 Fluorometer (Invitrogen), (unrelated healthy control subjects) were recruited from diluted to 4 nmol/L, and denatured with 0.2 N NaOH at university employees and children of employees. The pres- room temperature. The denatured DNA was diluted to ence of antibodies against GAD65, insulin, ICA512, and 15 pmol/L and spiked with 25% of the Illumina PhiX Con- ZnT8 was determined by the Autoantibody/HLA Service trol DNA prior to loading the sequencer. Illumina paired-end Center at the Barbara Davis Center for Diabetes. Typing sequencing was performed on the MiSeq platform with for HLA DR3 and DR4 of study subjects was performed by version 2.3.0.8 of the MiSeq Control Software and version PCR analysis (18). Thirty-two of the 35 new-onset patients 2.3.32 of MiSeq Reporter, using a 600-cycle version 3 (91%) had DR3 and/or DR4 HLA diabetes risk alleles. All reagent kit. autoantibody seropositive subjects (21 of 21) and 29 of 32 As previously described (9), Illumina MiSeq paired-end seronegative FDRs carried DR3 and/or DR4 (91%). Twelve sequences were sorted by sample via barcodes in the out of the 23 unrelated healthy subjects had DR3 and/or paired reads with a python script. The sorted paired reads DR4 (52%). The median HbA1c level in the seropositive and were assembled using phrap (20,21). Pairs that did not new-onset subject cohorts was 5.0% (range 4.2–5.3) and assemble were discarded. Assembled sequence ends were Table 1—Cohort characteristics Unrelated healthy New-onset control subjects patients Seropositive Seronegative FDRs Variable (n = 23) (n = 35) (n = 21) (n = 32) Females/males, n/n 9/14 16/19 12/9 14/18 Age (years), median (range) 12 (4–24) 11 (2–20) 9 (4–49) 12 (3–45) HLA, n (%) 3 alone 5 (22) 10 (29) 5 (24) 11 (34) 4 alone 3 (13) 13 (37) 12 (57) 8 (25) 3 plus 4 4 (17) 9 (26) 4 (19) 10 (31) x/x 11 (48) 3 (8) 0 3 (10) Autoantibodies, n (%) Zero – 2 (6) 0 32 (100) One – 12 (34) 5 (24) 0 Two to four – 21 (60) 16 (76) 0 HbA1c, median (range) – 7.9 (5.5–15.0) 5.0 (4.2–5.3) – Age at onset (years), median (range) – 10 (2–20) –– Disease duration (weeks), median (range) – 5.1 (0.3–17.3) –– Impaired glucose metabolism, n (%) ––5 (24) – 3512 Gut Microbiota and Islet Autoimmunity Diabetes Volume 64, October 2015 trimmed over a moving window of five nucleotides until of 16S rRNA genes (primers 534F–805R [9,10]) from fe- average quality met or exceeded 20. Trimmed sequences cal samples of seropositive individuals with one to four with more than one ambiguity or shorter than 200 autoantibodies (n = 21) compared with seronegative FDRs nucleotides were discarded. Potential chimeras identified (n = 32). Comparisons were also made between the gut with Uchime (usearch6.0.203_i86linux32) (22) using the bacterial content of autoantibody-positive individuals and Schloss (23) Silva reference sequences were removed from seronegative FDRs to that of new-onset patients (n = 35) subsequent analyses. Assembled sequences were aligned and autoantibody-free healthy control subjects with no and classified with SINA (1.2.11) using the 629,124 bac- family history of autoimmunity (n = 23).