Diabetes Volume 63, February 2014 619 Aimon K. Alkanani,1 Naoko Hara,1 Egil Lien,2 Diana Ir,3 Cassandra V. Kotter,3 Charles E. Robertson,4,5 Brandie D. Wagner,5,6 Daniel N. Frank,3,5 and Danny Zipris1 Induction of Diabetes in the RIP-B7.1 Mouse Model Is Critically Dependent on TLR3 and MyD88 Pathways and Is Associated With Alterations IMMUNOLOGY AND TRANSPLANTATION in the Intestinal Microbiome RIP-B7.1 transgenic mice express B7.1 TLR9-deficient mice treated with antibiotics plus costimulatory molecules in pancreatic islets and poly I:C had higher bacterial diversity compared with develop diabetes after treatment with polyinosinic: disease-resistant mice. Furthermore, principal polycytidylic acid (poly I:C), a synthetic double- component analysis suggested that TLR9-deficient stranded RNA and agonist of Toll-like receptor (TLR) 3 mice had distinct gut microbiome compared with the and retinoic acid–inducible protein I. We used this diabetes-resistant mice. Finally, the administration model to investigate the role of TLR pathways and of sulfatrim plus poly I:C to TLR9-deficient mice intestinal microbiota in disease progression. RIP- resulted in alterations in the abundance of gut B7.1 mice homozygous for targeted disruption of bacterial communities at the phylum and genus TLR9, TLR3, and myeloid differentiation factor-88 levels. These data imply that the induction of (MyD88), and most of the wild-type RIP-B7.1 mice diabetes in the RIP-B7.1 model is critically housed under normal conditions remained diabetes- dependent on TLR3 and MyD88 pathways, and free after poly I:C administration. However, the involves modulation of the intestinal microbiota. majority of TLR9-deficient mice and wild-type Diabetes 2014;63:619–631 | DOI: 10.2337/db13-1007 animals treated with poly I:C and an antibiotic developed disease. In sharp contrast, TLR3- and MyD88-deficient mice were protected from diabetes Type 1 diabetes (T1D) is a proinflammatory autoimmune following the same treatment regimen. High- disease that involves the specific destruction of pancre- throughput DNA sequencing demonstrated that atic b-cells (1). The etiology of the disease is incompletely 1Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, 6Department of Biostatistics and Informatics, Colorado School of Public Health, Aurora, CO University of Colorado Denver, Aurora, CO 2Department of Medicine, Division of Infectious Diseases and Immunology, Corresponding author: Danny Zipris, [email protected]. University of Massachusetts Medical School, Worcester, MA Received 27 June 2013 and accepted 7 October 2013. 3Division of Infectious Diseases, University of Colorado School of Medicine, Aurora, CO This article contains Supplementary Data online at http://diabetes 4Department of Molecular, Cellular, and Developmental Biology, University of .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1007/-/DC1. Colorado, Boulder, CO © 2014 by the American Diabetes Association. See http://creativecommons 5University of Colorado Microbiome Research Consortium, Aurora, CO .org/licenses/by-nc-nd/3.0/ for details. 620 TLR Pathways in the RIP-B7.1 Mouse Diabetes Volume 63, February 2014 understood; however, epidemiological data and evidence The B7.1 transgene expression and/or gene mutation from animal models of T1D implicate microbes in the were detected by PCR amplification using genomic DNA disease course (2). isolated from tail snips (22). All animals were housed in The innate immune system uses pattern-recognition a specific pathogen-free facility and maintained in ac- receptors, such as Toll-like receptors (TLRs), nucleotide- cordance with the Guide for the Care and Use of Labo- binding oligomerization domain (NOD)-like receptors, ratory Animals (published by the National Institutes of and retinoic acid–inducible gene-1 (RIG-I)–like receptors, Health, NIH publication no. 86-23, revised 1996) and the to detect and eradicate microbial infections (3). Inter- guidelines of the Institutional Animal Care and Use actions between TLRs and their ligands initiate a series of Committee of the University of Colorado Denver. intracellular signaling events leading to the expression of fl TLR Activation, Antibiotic Treatment, Blood Removal, antimicrobial genes, proin ammatory cytokines, and and Serum p40 Measurements chemokines, such as type I interferons, interleukin (IL)-1, To induce diabetes, mice at 6–8 weeks of age were IL-6, IL-12, and chemokine (C-X-C motif) ligand-10 (3,4). injected intraperitoneally with 5 mg/g body weight poly I: Emerging evidence has implicated the innate immune C (Sigma-Aldrich, St. Louis, MO) on 10 consecutive days. system and TLR pathways in the mechanism of T1D Mice were given normal water or water supplemented (reviewed in Chervonsky [5], Wong and Wen [6], and with sulfatrim (1 mg/mL sulfamethoxazole plus 0.2 Zipris [7]). mg/mL trimethoprim; Hi-Tech Pharmacal, Amityville, The gut microbiota play a central role in shaping the NY) beginning at birth and continuing for the duration of peripheral and intestinal immune systems in health and the experiment. Animals were monitored for disease disease (5,8–10). The gut microbiome has recently been development for at least 200 days after poly I:C treat- implicated in chronic proinflammatory disorders, in- ment. Blood glucose was measured twice a week, and the cluding T1D (11–18). A temporal decline in members of mice were considered diabetic after two consecutive the bacterial phyla Firmicutes and an increase in Bac- blood glucose test results of .250 mg/dL. To investigate teroidetes has been observed in individuals that are at the expression of the p40 subunit of IL-12 and IL-23, risk for developing T1D, compared with healthy subjects mice received 5 mg/g body weight of poly I:C or CpG DNA (13). Other investigators have shown that intestinal (TriLink BioTechnologies). Blood was collected 6 h after bacteria are associated with T1D in the diabetes-prone the treatment. Sera were separated from blood cells im- BioBreeding rat (19), the LEW1.WR1 rat model of virus- mediately after blood collection and stored at 280°C induced T1D (20), and the NOD mouse (14,21). until use. The serum level of the p40 subunit of IL-12 The RIP-B7.1 transgenic mouse expresses B7.1 co- and IL-23 was measured using a commercial kit from Life stimulatory molecules under the control of the rat insulin Technologies-Invitrogen (Carlsbad, CA) according to the promoter (22). The administration of double-stranded manufacturer’s instructions. RNA, a ligand of TLR3 and RIG-like helicases, in these animals results in diabetes via mechanisms that have DNA Isolation and Quantitative RT-PCR Analysis for been hypothesized to involve type I interferon pathways the Detection of Gut Bacteria (23) and the upregulation of antigen-presenting cells The Lactobacillus, Bifidobacterium, Clostridium, and Bac- and T-cell auto-reactivity (24). In the current study, we teroides genera were detected in stool DNA by quantita- used the RIP-B7.1 mouse model of diabetes to investigate tive PCR analysis using previously published primers the role of TLR pathways and the intestinal microbiota in (26). Bacterial DNA was recovered from stool using the the mechanism of disease induction. We demonstrate QIAmp DNA stool mini kit (Qiagen, Valencia, CA) that the TLR3 and myeloid differentiation factor-88 according to the manufacturer’s instructions. DNA con- (MyD88) pathways, but not the TLR9 pathway, play centrations were determined using a NanoDrop ND-1000 a critical role in the mechanism of islet destruction. spectrophotometer (Thermo Scientific, Wilmington, DE). Furthermore, we provide evidence that alterations in the The data were normalized to the total bacterial DNA in gut microbiota may be involved in the course of poly- each sample using previously described primers and inosinic:polycytidylic acid (poly I:C)–induced diabetes in conditions (26). this animal model. High-Throughput DNA Sequencing for Microbiome RESEARCH DESIGN AND METHODS Analysis Mice 16S Amplicon Library Construction C57BL/6 mice with pancreatic b-cells expressing B7.1 Bacterial profiles from the mice were determined by costimulatory molecules under the rat insulin promoter broad-range amplification and sequence analysis of 16S (RIP-B7.1) were produced as previously described (22) ribosomal RNA (rRNA) genes following our previously and bred in the University of Colorado animal facility. To described methods (20,27). In brief, amplicons were generate RIP-B7.1 mice deficient in TLR9, TLR3, or generated using primers that target approximately 300 MyD88, RIP-B7.1 mice were bred to homozygosity with base pairs of the V4 variable region of the 16S rRNA C57BL/6 mice deficient in TLR9, TLR3, or MyD88 (25). gene. PCR products were normalized using a SequalPrep diabetes.diabetesjournals.org Alkanani and Associates 621 kit (Invitrogen), and were pooled, lyophilized, purified, component analysis (PCA) was performed using singular and concentrated using a DNA Clean and Concentrator value decomposition of the centered data using the Kit (Zymo, Irvine, CA). Pooled amplicons was quantified prcomp function in R. A small constant (1/total number using Qubit Fluorometer 2.0 (Invitrogen). The pool was of sequences) was added to the counts prior to the ap- diluted to 2 nmol/L and denatured with 0.2N NaOH at plication of the centered log ratio transformation rec- room temperature. The denatured DNA was diluted to 15 ommended for compositional data (35,36). pmol/L and spiked with 25% of the Illumina PhiX control DNA prior to loading the sequencer. Illumina paired-end RESULTS sequencing was performed on the Miseq platform with Poly I:C–Induced Diabetes in RIP-B7.1 Mice with version 2.0 of the Miseq Control Software, using a 500- Disrupted TLR Signaling cycle version 2 reagent kit. TLR pathways have been suggested to play a key role in the mechanism leading to diabetes in humans and in an Analysis of Illumina Paired-End Reads animal model (5,6,37,38).