Diabetes Volume 63, August 2014 2821 Camilla Hartmann Friis Hansen,1 Łukasz Krych,2 Karsten Buschard,3 Stine B. Metzdorff,1 Christine Nellemann,4 Lars H. Hansen,5 Dennis S. Nielsen,2 Hanne Frøkiær,1 Søren Skov,1 and Axel K. Hansen1 A Maternal Gluten-Free Diet Reduces Inflammation and Diabetes Incidence in the Offspring of NOD Mice Diabetes 2014;63:2821–2832 | DOI: 10.2337/db13-1612 Early-life interventions in the intestinal environment gluten-free (GF) diet decreased the incidence of diabetes have previously been shown to influence diabetes in- from 64% to 15% when nonobese diabetic (NOD) mice cidence. We therefore hypothesized that a gluten-free were fed a GF diet after weaning (1), and eating a GF diet (GF) diet, known to decrease the incidence of type 1 decreased the incidence of diabetes to just 6% in the off- PATHOPHYSIOLOGY diabetes, would protect against the development of spring in two generations, which indicates that the inter- diabetes when fed only during the pregnancy and lactation play between gut antigens and immune pathways leading period. Pregnant nonobese diabetic (NOD) mice were to diabetes is particularly important in the preweaning fed a GF or standard diet until all pups were weaned to period when insulitis starts to progress (2). a standard diet. The early-life GF environment dramat- Accumulating evidence suggests that gut immune ically decreased the incidence of diabetes and insulitis. reactivity is skewed in human and murine diabetic Gut microbiota analysis by 16S rRNA gene sequencing patients. Studies in young human patients with T1D revealed a pronounced difference between both moth- have demonstrated increased numbers of interferon-g ers and their offspring on different diets, characterized – – Akkermansia (IFN-g) producing, interleukin (IL)-1a producing, and by increased numbers of , Proteobacteria, – and TM7 in the GF diet group. In addition, pancreatic IL-4 producing cells in the small intestinal lamina propria, fl forkhead box P3 regulatory T cells were increased in re ecting T1D preceded by intestinal immune activation GF-fed offspring, as were M2 macrophage gene markers (3). Similarly in NOD mice, a diabetes-promoting diet fl and tight junction–related genes in the gut, while intesti- induced proin ammatory cytokines IFN-g and tumor ne- nal gene expression of proinflammatory cytokines was crosis factor-a in the small intestinal lamina propria (4), reduced. An increased proportion of T cells in the pan- and an antidiabetogenic diet decreased the high numbers + + creas expressing the mucosal integrin a4b7 suggests of CD11b CD11c dendritic cells (DCs) found in the colon that the mechanism involves increased trafficking of lamina propria (5). Under germ-free conditions, reduced gut-primed immune cells to the pancreas. In conclusion, expression of forkhead box P3 (FoxP3) in the ileum, colon, a GF diet during fetal and early postnatal life reduces and the draining lymph node was associated with acceler- the incidence of diabetes. The mechanism may involve ated development of insulitis in NOD mice (6), and, likewise changes in gut microbiota and shifts to a less proinflam- in humans, Badami et al. (7) found that jejunal biopsy matory immunological milieu in the gut and pancreas. samples from T1D patients showed reduced frequency of 2 CD4+CD25+FoxP3+CD127 regulatory T cells (Tregs). The Gluten has previously been shown to affect the devel- link between the gut and pancreas has also been empha- opment of type 1 diabetes (T1D) in animal models. A sized in studies demonstrating that pancreatic islet T cells 1Department of Veterinary Disease Biology, Faculty of Health and Medical Sci- 5Department of Biology, Faculty of Science, University of Copenhagen, Copenha- ences, University of Copenhagen, Frederiksberg, Denmark gen, Denmark 2 Department of Food Science, Faculty of Science, University of Copenhagen, Corresponding author: Camilla Hartmann Friis Hansen, [email protected]. Frederiksberg, Denmark Received 18 October 2013 and accepted 27 March 2014. 3Bartholin Institute, Rigshospitalet, Copenhagen, Denmark 4Division of Toxicology and Risk Assessment, National Food Institute, Technical © 2014 by the American Diabetes Association. Readers may use this article as University of Denmark, Søborg, Denmark long as the work is properly cited, the use is educational and not for profit, and the work is not altered. 2822 Diabetes-Protective Diet Across Two Generations Diabetes Volume 63, August 2014 express gut homing receptor a4b7 integrin, which recog- at 4 weeks of age to the STD diet. Ten pups were killed nizes mucosal addressin cell adhesion molecule-1 in the from each group at 4 weeks of age, and 10 mice from each pancreas (8,9). group were killed at 10 weeks of age. The remaining 30 Failure in immune tolerance leading to pancreatic mice in each group were killed when a diagnosis of di- b-cell depletion has been suggested to be regulated in abetes was made or at 30 weeks of age, when the study part by gliadin-induced intestinal enteropathy and innate ended. Measurements of tail blood glucose levels were immune responses (10,11). However, altered gut micro- made twice a week from 10 weeks of age, and a mouse biota, previously demonstrated in GF-fed versus gluten- was considered to be diabetic when blood glucose levels fed mice (12), might also contribute to modify intestinal exceeded 12 mmol/L on 2 consecutive days. The body inflammation and development of autoimmune diabetes. weight of all offspring was monitored once a week. In support of this, impaired oral tolerance to intestinal Histology microbes was demonstrated in NOD mice (5), and the Hematoxylin-eosin–stained pancreas sections were evalu- fi impact of microbes has also been veri ed in germ-free ated for insulitis score in a blinded fashion by two per- (13), antibiotic-treated (14,15), and probiotic-treated di- sons. Lymphocytic infiltration was graded as follows: 0, abetes-prone rodent models (16). It seems reasonable to no infiltration; 1, intact islets but with few mononuclear assume that gluten and certain microbes have a synergistic cells surrounding the islets; 2, peri-insulitis; 3, islet in- effect on the development of T1D, as was also recently filtration ,50%; and 4, islet infiltration .50%. Twenty- fi suggested by Patrick et al. (17). Cytokine pro les of gut- five islets were scored for each nondiabetic mouse killed at associated lymphoid tissue have revealed a strong associa- 10 and 30 weeks of age. tion between intestinal IFN-g production and the incidence of diabetes, especially in several gluten intervention stud- Gut Microbiota ies (4,11,17–20). Also, type 1 T-helper cells proliferated Feces samples aseptically obtained from the mothers specifically in the mesenteric lymph node (MLN) in re- during pregnancy and from the offspring at 4 and 10 sponse to wheat protein antigens (19). A GF diet was weeks of age when they were killed were analyzed by PCR fi furthermore shown to reverse this shift in gut homeosta- ampli cation of the V3 region of the 16S rRNA gene foll- sis toward an anti-inflammatory state with more trans- owed by denaturing gradient gel electrophoresis (DGGE) fi forming growth factor-b (TGF-b)–producing T cells (18). as described previously (21). The resulting DGGE pro les As early-life interventions in the intestinal environ- were analyzed using BioNumerics version 4.5 (Applied ment can influence the incidence of diabetes, we hypoth- Maths, Sint-Martens-Latem, Belgium). The composition esized that a GF diet exclusively fed to mice during of the prokaryotic community of feces samples from gestation and lactation would be sufficient to protect the the mothers and the 4-week-old pups was determined offspring from the development of diabetes even though using tag-encoded 454/FLX Titanium (Roche) pyrose- they were weaned to a standard gluten-containing (STD) quencing of the V3 and V4 regions of the 16S rRNA diet. We hypothesized that the dietary protective effect gene by the National High Throughput DNA Sequen- would be partly mediated by a shift in the gut microbiota, cing Centre, University of Copenhagen, Copenhagen, and that this shift is of imperative importance in the first Denmark (22), and was analyzed as described by Krych period of life during which the immune system develops. et al. (23). An open source software package, Quantitative Insight Into Microbial Ecology (QIIME version 1.7.0) was RESEARCH DESIGN AND METHODS used to analyze the pyrosequencing data (National Cen- ter for Biotechnology Information database accession The experiment was performed in accordance with the #PRJNA215143). Principal coordinate analysis (PCoA) Council of Europe Convention European Treaty Series was made using the jackknife_beta_diversity.py workflow 123 on the Protection of Vertebrate Animals used for (the –e value: 2,000 sequences). The PCoA plot including Experimental and Other Scientific Purposes, and the bacterial taxa was drawn out with the make_3d_plots.py Danish Animal Experimentation Act (LBK 1306 from 23 script based on the summary information of bacterial November 2007). The study was approved by the Animal phyla, and the differences in the taxa relative distribution Experiments Inspectorate, Ministry of Justice, Denmark. between categories were tested with Metastats (24) Animals and Diet independently for both the phylum-level and the genus- NOD/BomTac mice (Taconic, Hudson, NY) were fed ad level summarized taxa. The P value was calculated based libitum either a GF modified Altromin diet or an STD on 1,000 permutations. Altromin diet (Altromin, Lage, Germany), as described by Cell Isolation and Flow Cytometry Funda et al. (1). The two groups were mated separately, Single-cell suspensions from spleen, MLN, and pancreatic and their female offspring were group-housed (five mice/ lymph node (PLN) isolated from 4- and 10-week-old cage) in our barrier-protected rodent facility (Faculty of offspring immediately upon their being killed, and flow Health and Medical Sciences, University of Copenhagen, cytometric analyses of CD11b+CD11c+ DCs and T-cell Frederiksberg, Denmark) under standard conditions in populations, including FoxP3+ Tregs, were performed as open cages without filter lids.