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 DietReduces Inflammation andDiabetes Incidence in theOffspring of NOD MiceDiabetes 2014;63:2821–2832 | DOI: 10.2337/db13-1612

Early-life interventions in the intestinal environmenthave previously been shown to influence diabetes in-cidence. We therefore hypothesized that a gluten-free(GF) diet, known to decrease the incidence of type 1diabetes, would protect against the development ofdiabetes when fed only during the pregnancy and lactationperiod. Pregnant nonobese diabetic (NOD) mice werefed a GF or standard diet until all pups were weaned toa standard diet. The early-life GF environment dramat-ically decreased the incidence of diabetes and insulitis.Gut microbiota analysis by 16S rRNA gene sequencingrevealed a pronounced difference between both moth-ers and their offspring on different diets, characterizedby increased numbers of Akkermansia, Proteobacteria,and TM7 in the GF diet group. In addition, pancreaticforkhead box P3 regulatory T cells were increased inGF-fed offspring, as were M2 macrophage gene markersand tight junction–related genes in the gut, while intesti-nal gene expression of proinflammatory cytokines wasreduced. An increased proportion of T cells in the pan-creas expressing the mucosal integrin a4b7 suggeststhat the mechanism involves increased trafficking ofgut-primed immune cells to the pancreas. In conclusion,a GF diet during fetal and early postnatal life reducesthe incidence of diabetes. The mechanism may involvechanges in gut microbiota and shifts to a less proinflam-matory immunological milieu in the gut and pancreas.

Gluten has previously been shown to affect the devel-opment of type 1 diabetes (T1D) in animal models. A

gluten-free (GF) diet decreased the incidence of diabetesfrom 64% to 15% when nonobese diabetic (NOD) micewere fed a GF diet after weaning (1), and eating a GF dietdecreased the incidence of diabetes to just 6% in the off-spring in two generations, which indicates that the inter-play between gut antigens and immune pathways leadingto diabetes is particularly important in the preweaningperiod when insulitis starts to progress (2).

Accumulating evidence suggests that gut immunereactivity is skewed in human and murine diabeticpatients. Studies in young human patients with T1Dhave demonstrated increased numbers of interferon-g(IFN-g)–producing, interleukin (IL)-1a–producing, andIL-4–producing cells in the small intestinal lamina propria,reflecting T1D preceded by intestinal immune activation(3). Similarly in NOD mice, a diabetes-promoting dietinduced proinflammatory cytokines IFN-g and tumor ne-crosis factor-a in the small intestinal lamina propria (4),and an antidiabetogenic diet decreased the high numbersof CD11b+CD11c+ dendritic cells (DCs) found in the colonlamina propria (5). Under germ-free conditions, reducedexpression of forkhead box P3 (FoxP3) in the ileum, colon,and the draining lymph node was associated with acceler-ated development of insulitis in NOD mice (6), and, likewisein humans, Badami et al. (7) found that jejunal biopsysamples from T1D patients showed reduced frequency ofCD4+CD25+FoxP3+CD1272 regulatory T cells (Tregs). Thelink between the gut and pancreas has also been empha-sized in studies demonstrating that pancreatic islet T cells

1Department of Veterinary Disease Biology, Faculty of Health and Medical Sci-ences, University of Copenhagen, Frederiksberg, Denmark2Department of Food Science, Faculty of Science, University of Copenhagen,Frederiksberg, Denmark3Bartholin Institute, Rigshospitalet, Copenhagen, Denmark4Division of Toxicology and Risk Assessment, National Food Institute, TechnicalUniversity of Denmark, Søborg, Denmark

5Department of Biology, Faculty of Science, University of Copenhagen, Copenha-gen, Denmark

Corresponding author: Camilla Hartmann Friis Hansen, camfriis@sund.ku.dk.

Received 18 October 2013 and accepted 27 March 2014.

© 2014 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

Diabetes Volume 63, August 2014 2821

PATHOPHYSIO

LOGY

express gut homing receptor a4b7 integrin, which recog-nizes mucosal addressin cell adhesion molecule-1 in thepancreas (8,9).

Failure in immune tolerance leading to pancreaticb-cell depletion has been suggested to be regulated inpart by gliadin-induced intestinal enteropathy and innateimmune responses (10,11). However, altered gut micro-biota, previously demonstrated in GF-fed versus gluten-fed mice (12), might also contribute to modify intestinalinflammation and development of autoimmune diabetes.In support of this, impaired oral tolerance to intestinalmicrobes was demonstrated in NOD mice (5), and theimpact of microbes has also been verified in germ-free(13), antibiotic-treated (14,15), and probiotic-treated di-abetes-prone rodent models (16). It seems reasonable toassume that gluten and certain microbes have a synergisticeffect on the development of T1D, as was also recentlysuggested by Patrick et al. (17). Cytokine profiles of gut-associated lymphoid tissue have revealed a strong associa-tion between intestinal IFN-g production and the incidenceof diabetes, especially in several gluten intervention stud-ies (4,11,17–20). Also, type 1 T-helper cells proliferatedspecifically in the mesenteric lymph node (MLN) in re-sponse to wheat protein antigens (19). A GF diet wasfurthermore shown to reverse this shift in gut homeosta-sis toward an anti-inflammatory state with more trans-forming growth factor-b (TGF-b)–producing T cells (18).

As early-life interventions in the intestinal environ-ment can influence the incidence of diabetes, we hypoth-esized that a GF diet exclusively fed to mice duringgestation and lactation would be sufficient to protect theoffspring from the development of diabetes even thoughthey were weaned to a standard gluten-containing (STD)diet. We hypothesized that the dietary protective effectwould be partly mediated by a shift in the gut microbiota,and that this shift is of imperative importance in the firstperiod of life during which the immune system develops.

RESEARCH DESIGN AND METHODS

The experiment was performed in accordance with theCouncil of Europe Convention European Treaty Series123 on the Protection of Vertebrate Animals used forExperimental and Other Scientific Purposes, and theDanish Animal Experimentation Act (LBK 1306 from 23November 2007). The study was approved by the AnimalExperiments Inspectorate, Ministry of Justice, Denmark.

Animals and DietNOD/BomTac mice (Taconic, Hudson, NY) were fed adlibitum either a GF modified Altromin diet or an STDAltromin diet (Altromin, Lage, Germany), as described byFunda et al. (1). The two groups were mated separately,and their female offspring were group-housed (five mice/cage) in our barrier-protected rodent facility (Faculty ofHealth and Medical Sciences, University of Copenhagen,Frederiksberg, Denmark) under standard conditions inopen cages without filter lids. All offspring were weaned

at 4 weeks of age to the STD diet. Ten pups were killedfrom each group at 4 weeks of age, and 10 mice from eachgroup were killed at 10 weeks of age. The remaining 30mice in each group were killed when a diagnosis of di-abetes was made or at 30 weeks of age, when the studyended. Measurements of tail blood glucose levels weremade twice a week from 10 weeks of age, and a mousewas considered to be diabetic when blood glucose levelsexceeded 12 mmol/L on 2 consecutive days. The bodyweight of all offspring was monitored once a week.

HistologyHematoxylin-eosin–stained pancreas sections were evalu-ated for insulitis score in a blinded fashion by two per-sons. Lymphocytic infiltration was graded as follows: 0,no infiltration; 1, intact islets but with few mononuclearcells surrounding the islets; 2, peri-insulitis; 3, islet in-filtration ,50%; and 4, islet infiltration .50%. Twenty-five islets were scored for each nondiabetic mouse killed at10 and 30 weeks of age.

Gut MicrobiotaFeces samples aseptically obtained from the mothersduring pregnancy and from the offspring at 4 and 10weeks of age when they were killed were analyzed by PCRamplification of the V3 region of the 16S rRNA gene foll-owed by denaturing gradient gel electrophoresis (DGGE)as described previously (21). The resulting DGGE profileswere analyzed using BioNumerics version 4.5 (AppliedMaths, Sint-Martens-Latem, Belgium). The compositionof the prokaryotic community of feces samples fromthe mothers and the 4-week-old pups was determinedusing tag-encoded 454/FLX Titanium (Roche) pyrose-quencing of the V3 and V4 regions of the 16S rRNAgene by the National High Throughput DNA Sequen-cing Centre, University of Copenhagen, Copenhagen,Denmark (22), and was analyzed as described by Krychet al. (23). An open source software package, QuantitativeInsight Into Microbial Ecology (QIIME version 1.7.0) wasused to analyze the pyrosequencing data (National Cen-ter for Biotechnology Information database accession#PRJNA215143). Principal coordinate analysis (PCoA)was made using the jackknife_beta_diversity.py workflow(the –e value: 2,000 sequences). The PCoA plot includingbacterial taxa was drawn out with the make_3d_plots.pyscript based on the summary information of bacterialphyla, and the differences in the taxa relative distributionbetween categories were tested with Metastats (24)independently for both the phylum-level and the genus-level summarized taxa. The P value was calculated basedon 1,000 permutations.

Cell Isolation and Flow CytometrySingle-cell suspensions from spleen, MLN, and pancreaticlymph node (PLN) isolated from 4- and 10-week-oldoffspring immediately upon their being killed, and flowcytometric analyses of CD11b+CD11c+ DCs and T-cellpopulations, including FoxP3+ Tregs, were performed aspreviously described (22). All antibodies were purchased

2822 Diabetes-Protective Diet Across Two Generations Diabetes Volume 63, August 2014

from eBiosciences (San Diego, CA). The analyses were per-formed using an Accuri C6 flow cytometer (Accuri Cytom-eters Inc., Ann Arbor, MI).

Quantitative PCRImmediately after the mice were killed, 1-cm fragments ofthe ileum and colon were placed in RNAlater (Ambion,Austin, TX), after all luminal content was scraped out ofthe gut. Homogenization, RNA isolation with MagMAX-96 RNA Isolation Kit (Ambion), and cDNA synthesis usingthe High-Capacity cDNA Reverse Transcriptase Kit (Ap-plied Biosystems, Foster City, CA) were performed asdescribed previously (25). An inventoried TaqMan MouseImmune Array (Appled Biosystems) containing 90 TaqMangene expression assays of immune-related genes was usedto investigate ileal samples isolated at weaning, which wereanalyzed as described previously (26). Actinb, Ocln, Tjp1,Cldn8, Cldn15, Muc1, and Muc2 TaqMan gene expressionassays (Applied Biosystems) were used for quantitativePCR (qPCR) analyses on ileum and colon cDNA isolatedat 4 weeks of age, and the data were analyzed as describedpreviously (25).

cDNA samples from the ileum and colon collectedat weaning were further analyzed for the presence ofAkkermansia muciniphila, which was quantified in dupli-cate using the 7500 Fast Real-time PCR System (AppliedBiosystems), as previously described (27).

Statistical AnalysisGraphPad Prism version 5.02 (GraphPad Software, SanDiego, CA) was used for statistical analysis, and P values,0.05 were considered to be significant. Cumulative di-abetes incidence was calculated using the Kaplan-Meierestimation, whereas statistical significance was evaluatedby the log-rank test. Other differences were estimatedby two-tailed t test or one-way ANOVA test with Tukeypost test.

RESULTS

A Maternal GF Diet Attenuates Diabetes in theOffspringIn this study, it was demonstrated that feeding a GF dietto pregnant NOD mice significantly reduced the cumu-lative diabetes incidence (P , 0.01) and increased theonset time (P , 0.01) in their offspring, even though allpups were weaned to an STD diet at 4 weeks of age (Fig.1A). The diabetes incidence at 210 days was 51% (n = 37)in the offspring of STD diet–fed mice, and 22% (n = 36)in the offspring of GF diet–fed mice. Histological evalu-ation of insulitis in pancreatic sections from nondiabeticoffspring revealed a significantly lower insulitis score inoffspring of GF diet–fed mice compared with the off-spring of STD diet–fed mice at both 10 weeks (P ,0.05; Fig. 1B and C) and 30 weeks of age (P , 0.05;Fig. 1D and E). No significant difference in body weightgain was observed between the two groups of nondia-betic NOD mice within the observational period (datanot shown).

GF Diet Leads to a Gut Microbiota Enriched inVerrucomicrobia, Proteobacteria, and TM7 in Damsand OffspringGut microbiota analysis by DGGE demonstrated a differ-ence in the fecal gut microbiota between the two groupsof pregnant NOD mice (Fig. 2A). ANOVA based on thefirst (X), second (Y), and third (Z) principal component(PC) revealed a significant difference in PC2 values (P ,0.05), and a tendency to cluster in PC1 (P = 0.07) and PC3values (P = 0.08). The separate clustering on the PC anal-ysis plot was also evident in their offspring at weaning,confirming that parental microbiomes altered by diet areinheritable (28); significant differences in PC1 values (P,

Figure 1—A: Cumulative diabetes incidence in female NOD off-spring of mice fed an STD diet (n = 37, blue) or a GF diet (n = 36,red). All offspring were weaned to an STD diet at 4 weeks of age andwere diagnosed as diabetic and killed when blood glucose levelsexceeded 12 mmol/L on 2 consecutive days. Comparisons of thetwo survival curves were tested by log-rank test, and the P value isshown. B: Average insulitis score for offspring of STD and GF diet–fed NOD mice at 10 weeks of age that were weaned to an STD diet.C: Percentage of islets with a given score in 10-week-old offspringof STD diet–fed (n = 9) and GF diet–fed (n = 8) NOD mice. D:Average insulitis score for nondiabetic offspring of STD and GFdiet–fed NOD mice at 30 weeks of age. E: Percentage of isletswith a given score in 30-week-old nondiabetic offspring of STDdiet–fed (n = 8) and GF diet–fed (n = 10) NOD mice. White, noinfiltration; light gray, few mononuclear cells infiltrated; gray, peri-insulitis; dark gray, <50% islet infiltration; black, >50% islet infil-tration. Error bars represent the SEM. *P < 0.05, **P < 0.01.

diabetes.diabetesjournals.org Hansen and Associates 2823

Figure 2—A: PC analysis plot based on DGGE profiles of 16S rRNA gene PCR–derived amplicons of feces samples collected from NODoffspring of mice fed an STD diet (n = 16, dark blue) or a GF diet (n = 16, red) at 4 weeks of age. STD diet–fed mothers (n = 6, light blue) andGF diet–fed mothers (n = 5, yellow) are also illustrated. B: PC analysis plot based on DGGE of feces collected from the two groups ofoffspring at 10 weeks of age, which were weaned to an STD diet at 4 weeks of age. ANOVA based on the X, Y, and Z PC analysis was usedto compare the groups. C: PCoA plot of 16S rRNA gene tag–encoded pyrosequencing reads based on the weighted UniFrac distancematrix showing clustering of the two groups of offspring. The gray spherical coordinates indicating bacterial phyla are plotted as a weightedaverage of sample coordinates. The size of each sphere is proportional to the mean relative abundance among all plotted samples. D: The

2824 Diabetes-Protective Diet Across Two Generations Diabetes Volume 63, August 2014

0.05) and in PC3 values (P, 0.001) were evident betweenthe two groups of offspring. However, how much of thisdifference in microbiota is due to vertical transfer frommothers to pups or to early ingestion of the GF diet by thepups is not known. Gut microbiota analysis of feces fromoffspring at 10 weeks of age, 6 weeks after weaning to theSTD diet, revealed no difference between the two groupsof offspring (Fig. 2B). The influence of the diet and thegut microbiota of the mothers on the gut microbiota ofthe offspring was thus not permanent.

The differences in the fecal microbial compositionbetween the two groups of NOD mice and their offspringat 4 weeks of age were further corroborated by tag-encoded 16S rRNA gene–based pyrosequencing. The rawnumber of reads generated from all 39 fecal samplesscored 1,332,137. Sequences that met all requirementsof the quality control (minimum length 300 bp, qualityscore $25) and were free from chimeric reads yielded848,346, providing an average of 21,752 sequences persample (minimum 1,178 sequences, maximum 88,674sequences, SD = 17,103 sequences), with a mean sequencelength of 458 bp (minimum 300 bp, maximum 470 bp).One sample was discarded because of the low number ofreads (,1,000 reads). PCoA based on weighted UniFracdistance metrics showed a clear separation of the twocategories comprising GF diet–fed NOD mice and theiroffspring, and STD diet–fed NOD mice with their off-spring. The proportion of the cumulative information de-scribing the variance using the first two PCs reached 60%(Fig. 2C).

The most abundant phyla in both categories wereFirmicutes and Bacteroidetes that constituted 50% and40%, respectively (Table 1). Metastats analysis revealed

that the difference observed between the groups was ma-inly due to a significantly expanded representation of thebacterial phyla Verrucomicrobia, TM7, and Proteobacteriain the mothers that were eating a GF diet and their pupscompared with STD diet–fed mice (Fig. 2D). In addition,the phylum Cyanobacteria was found in approximatelyhalf of the offspring of STD diet–fed mice, but in noneof the offspring of GF diet–fed mice. The annotation ofreads within the Verrucomicrobia represented one spe-cies, A. muciniphila, whereas the genus Proteus was respon-sible for the difference evident in Proteobacteria phyla(Table 2).

qPCR analysis of Akkermansia in the ileum mucosallayer at weaning revealed a higher abundance in the off-spring of GF diet–fed mice compared with STD diet–fedmice (Fig. 2E; P , 0.001). A similar tendency was alsoevident in the colonic mucus layer (Fig. 2F; P = 0.06).An Early GF Environment Increases Anti-InflammatoryImmune Cells and Intestinally Primed T Cells in PLNBoth intestinal and pancreatic DCs have been reported toplay important roles in T1D, and especially the CD11b+

subset has been associated with both pathogenic and tol-erogenic immunity to pancreatic islets. In this study, theCD11b+CD11c+ DCs were less abundant among pancreatic(P, 0.01), intestinal (P, 0.001), and systemic (P, 0.05)lymphocyte populations in the offspring of GF diet–fedmice compared with STD diet–fed offspring at the timeof weaning (Fig. 3A). No differences in the overall propor-tion of CD11c+ DCs were observed, and no differences wereobserved between the groups at 10 weeks of age.

The proportions of Tregs (CD4+FoxP3+) in the MLNand spleen were similar in the offspring of GF diet–fedand STD diet–fed mice, both at weaning and at 10 weeks

Table 1—Phyla relative distribution in feces samples collected from 4-week-old offspring of STD diet–fed and GF diet–fed NODmice

Taxa STD diet mean abundance GF diet mean abundance P value* q value*

Cyanobacteria 1.276 0.001 0.001 0.009

Verrucomicrobia 0.686 7.811 0.002 0.016

Proteobacteria 0.176 0.969 0.001 0.016

TM7 0.012 6.062 0.001 0.009

Firmicutes 54.510 49.030 0.501 1.000

Bacteroidetes 42.545 35.747 0.362 1.000

Deferribacteres 0.544 0.055 0.029 0.139

Tenericutes 0.057 0.007 0.195 0.669

Actinobacteria 0.005 0.000 0.086 0.407

*Values were calculated with Metastats using 1,000 permutations.

relative distribution of bacterial phyla across samples. Bar charts represent the relative abundance of the seven major phyla acrosssamples. Taxonomy assignation was based on the Greengenes reference database (version 12_10). E: qPCR analysis of A. muciniphila inileum samples from 4-week-old NOD offspring of mice fed an STD diet (n = 8) or a GF diet (n = 8). F: qPCR analysis of A. muciniphila inileum samples from 4-week-old NOD offspring of mice fed an STD diet (n = 9) or a GF diet (n = 9). The relative distribution of A. muciniphilawithin all bacteria is shown for each sample. All samples analyzed by qPCR were quantified in duplicate.

diabetes.diabetesjournals.org Hansen and Associates 2825

Tab

le2—

Significa

ntdifferen

cesin

taxa

among

4-wee

k-old

offsp

ring

ofSTD

diet–fedan

dGFdiet–fedNOD

mice

Phy

lum

Class

Order

Family

Gen

usSTD

diet

mea

nab

undan

ceGFdietmea

nab

undan

cePva

lue*

qva

lue*

Firm

icutes

Clostrid

iaUnc

lass

ified

Unc

lass

ified

Unc

lassified

0.46

01.81

80.00

10.01

1

Firm

icutes

Clostrid

iaUnk

nown

Unk

nown

Unk

nown

0.12

81.61

60.00

10.01

1

Firm

icutes

Clostrid

iaClostrid

iales

Unc

lassified

Unc

lassified

0.09

60.35

80.00

10.01

1

Firm

icutes

Clostrid

iaClostrid

iales

Halob

acteria

ceae

Deh

alob

acteriu

m0.12

50.61

40.00

10.01

1

Firm

icutes

Clostrid

iaClostrid

iales

Lach

nosp

irace

aeRos

eburia

0.00

00.10

40.00

10.01

1

Firm

icutes

Clostrid

iaClostrid

iales

Lach

nosp

irace

ae[Rum

inoc

occu

s]0.15

30.59

40.00

10.01

1

Firm

icutes

Clostrid

iaClostrid

iales

Rum

inoc

occa

ceae

Unc

lassified

0.22

50.95

00.00

10.01

1

Firm

icutes

Clostrid

iaClostrid

iales

Rum

inoc

occa

ceae

Unk

nown

0.28

91.44

60.00

30.02

6

Firm

icutes

Clostrid

iaClostrid

iales

Rum

inoc

occa

ceae

Ana

erotrunc

us0.04

80.18

10.00

10.01

1

Firm

icutes

Clostrid

iaClostrid

iales

Rum

inoc

occa

ceae

Osc

illos

pira

5.84

411

.871

0.00

60.04

9

Proteob

acteria

Gam

map

roteob

acteria

Enterob

acteria

les

Enterob

acteria

ceae

Proteus

0.00

00.18

70.00

20.02

0

TM7

TM7-3

CW04

0F1

6Unk

nown

0.01

26.05

70.00

10.01

1

Verruco

microbia

Verruco

microbiae

Verruco

microbiales

Verruco

microbiace

aeAkkerman

sia

0.68

67.80

90.00

30.02

6

Bac

teroidetes

Bac

teroidia

Bac

teroidales

[Parap

revo

tellace

ae]

[Prevo

tella]

1.91

80.01

20.00

10.01

1

Cya

nobac

teria

4C0d

-2YS2

Unk

nown

Unk

nown

1.25

50.00

00.00

10.01

1

Firm

icutes

Bac

illi

Lactob

acillales

Lactob

acillac

eae

Unk

nown

0.43

70.02

50.00

10.01

1

Firm

icutes

Clostrid

iaClostrid

iales

Unk

nown

Unk

nown

1.51

70.16

70.00

20.02

0

Firm

icutes

Clostrid

iaClostrid

iales

Clostrid

iace

aeUnc

lassified

0.96

40.00

10.00

10.01

1

Brack

etsindicatesu

gges

tedbut

notve

rified

names

.Unc

lass

ified

,inab

ility

toas

sign

give

nop

erationa

ltax

onom

icun

it(OTU

)intosing

letaxo

nomic

grou

p;Unk

nown,

abse

nceof

inform

ation

abou

tgive

nOTU

inthedatab

ase.

*Value

swereca

lculated

with

Metas

tats

using1,00

0permutations

.

2826 Diabetes-Protective Diet Across Two Generations Diabetes Volume 63, August 2014

of age, but, interestingly, a significantly higher proportion(P , 0.001) of these cells was evident in the PLN in theoffspring of GF diet–fed mice compared with offspring ofSTD diet–fed mice at weaning, indicating a more anti-in-flammatory local immune system due to an early GF en-vironment (Fig. 3B). The proportions of CD4 and CD8

T cells in the MLN, PLN, and spleen were not differentbetween the two groups either at weaning or at 10 weeksof age, except for a higher proportion (P , 0.001) of CD8T cells in the PLN at weaning in the offspring of GF diet–fed mice compared with offspring of STD diet–fed mice(Fig. 3G and H). Furthermore, these CD8 T cells were

Figure 3—Flow cytometric analysis of cells isolated from PLN, MLN, and spleen. Percentages of CD11b+CD11c+ DCs (A) and FoxP3+CD4+

Tregs (B) isolated from 4-week-old offspring from GF diet–fed (n = 8) or STD diet–fed (n = 8) NOD mice. C and D: Representative flowcytometric dot plots illustrating the percentages of CD11b+CD11c+ DCs in MLN after gating on CD11c+ cells. E and F: Representative flowcytometric dot plots illustrating the percentages of FoxP3+CD4+ Tregs in PLN after gating on CD4+ T cells. Percentages of CD8+ T cells (G),CD4+ T cells (H), a4b7+CD8+ T cells (I), and a4b7+CD4+ T cells (J) isolated from 4-week-old offspring from GF diet–fed (n = 8) or STD diet–fed (n = 8) NOD mice are illustrated as indicated. K–N: Representative flow cytometric dot plots illustrating the percentages of a4b7+CD8+

and a4b7+CD4+ T cells in PLN after gating on CD8+ and CD4+ T cells, respectively, as indicated. Error bars represent the SEM. *P < 0.05,**P < 0.01, ***P < 0.001.

diabetes.diabetesjournals.org Hansen and Associates 2827

marked with gut-homing receptor a4b7 integrin, which isinduced when T cells are activated in the intestinal envi-ronment (Fig. 3I). Also on the CD4 T cells in PLN a higherproportion of cells (P , 0.01) was marked with a4b7 inthe offspring of GF diet–fed mice compared with off-spring of STD diet–fed mice (Fig. 3J). However, this dif-ference in a4b7 integrin was evident only at weaning andnot at 10 weeks of age.

Intestinal Gene Expression Is Skewed Toward anAnti-Inflammatory Phenotype in Offspring of GFDiet–Fed MiceTo further explore how the early-life GF environ-ment affected gut homeostasis and the establishment of

regulatory immunological activity, genes implicated inimmune cell migration, microbial recognition, and re-sponse, as well as T-cell activation and signaling in the gutwere analyzed at weaning. Thirty-one of the 90 geneexpressions analyzed were significantly altered betweenthe two groups of offspring (Table 3). Of importance, theearly GF environment caused significantly less expressionof several inflammatory mediators of insulitis such asIfng, Il12b, and Il18 genes, and Prf1 and Gzmb genes forthe cytolytic enzymes perforin-1 and granzyme B, respec-tively. Interestingly, the Cd68 macrophage marker, theHmox1 gene for the heme oxygenase-1 enzyme, and theStat6 gene involved in exerting IL-4 were more

Table 3—Relative gene expression of immune-related genes analyzed in an inventoried TaqMan Mouse Immune Array in ilealsamples collected from 4-week-old offspring of GF diet–fed and STD diet–fed NOD mice

Gene Gene name [protein name]

RQ mean*

P value†STD diet GF diet

SignalingC3 Complement component 3 [C3] 1.07 6 0.14 2.06 6 0.33 ,0.01Col4a5 Procollagen, type IV, a5 [COL4a5] 1.06 6 0.16 2.63 6 0.66 ,0.05Edn1 Endothelin 1 [EDN1] 1.28 6 0.39 6.65 6 1.12 ,0.001Ikbkb Inhibitor of kB kinase b [IKKb] 1.03 6 0.09 1.51 6 0.17 ,0.05Nfkb1 Nuclear factor of k light chain gene enhancer in

B-cells 1 [NFkB1]1.02 6 0.07 1.37 6 0.12 ,0.05

Smad3 MAD homolog 3 (Drosophila) [SMAD3] 1.03 6 0.09 1.67 6 0.11 ,0.001Smad7 MAD homolog 7 (Drosophila) [SMAD7] 1.03 6 0.09 1.64 6 0.16 ,0.01Ski Sloan-Kettering viral oncogene homolog [SKI] 1.01 6 0.05 1.79 6 0.12 ,0.0001Socs1 Suppressor of cytokine signaling 1 [SOCS1] 1.03 6 0.10 0.51 6 0.11 ,0.01Socs2 Suppressor of cytokine signaling 2 [SOCS2] 1.02 6 0.07 1.89 6 0.17 ,0.001Stat6 Signal transducer and activator of

transcription 6 [STAT6]1.02 6 0.07 1.66 6 0.11 ,0.001

Cytokines/cytokine receptorsFas Fas (TNF receptor superfamily member) [FAS] 1.02 6 0.07 1.50 6 0.16 ,0.05Ifng IFN-g 3.13 6 1.63 0.29 6 0.22 ,0.01Il10 IL-10 1.25 6 0.35 0.00 6 0.00 ,0.0001Il12b IL-12b 3.42 6 1.55 0.85 6 0.80 ,0.05Il15 IL-15 1.06 6 0.15 3.05 6 0.45 ,0.0001Il17 IL-17 1.25 6 0.33 0.33 6 0.32 ,0.001Il18 IL-18 1.03 6 0.09 0.20 6 0.02 ,0.0001

Chemokine/chemokine receptorsCcr4 Chemokine (C-C motif) receptor 4 [CCR4] 1.72 6 0.37 0.15 6 0.04 ,0.01Cxcl11 Chemokine (C-X-C motif) ligand 11 [I-TAC] 1.03 6 0.11 3.18 6 0.56 ,0.0001

Cell surface receptorsCd34 CD34 antigen [CD34] 1.12 6 0.22 2.53 6 0.46 ,0.01Cd38 CD38 antigen [CD38] 1.01 6 0.06 0.52 6 0.06 ,0.01Cd3e CD3 antigen, ´ polypeptide [CD3e] 1.16 6 0.19 0.57 6 0.12 ,0.05Cd68 CD68 antigen [CD68] 1.04 6 0.11 1.56 6 0.20 ,0.05Cd8a CD8 antigen, a chain [CD8a] 1.19 6 0.23 0.49 6 0.10 ,0.05Lrp2 LDL receptor–related protein 2 [LRP2] 1.39 6 0.29 4.16 6 0.67 ,0.01Tfrc Transferrin receptor [TRFC] 1.02 6 0.08 1.38 6 0.11 ,0.05

EnzymesGzmb Granzyme B [CTLA1] 1.55 6 0.40 0.19 6 0.09 ,0.01Hmox1 Heme oxygenase (decycling) 1 [HMOX1] 1.09 6 0.19 2.41 6 0.48 ,0.05Nos2 Nitric oxide synthase 2, inducible, macrophage [NOS2] 1.05 6 0.12 0.15 6 0.04 ,0.0001Prf1 Perforin 1 (pore-forming protein) [PRF1] 1.77 6 0.47 0.44 6 0.20 ,0.05

*Relative quantification was calculated by the comparative Ct method, where the expression of each gene is first normalized to theexpression of Actb. Comparative gene expression is calculated for the mean control group, and fold change (RQ) values are obtained,with fold change = 1 for mean control. RQ values for genes more highly expressed compared with controls are marked in bold. †Onlygenes that were significantly expressed differently (P , 0.05) between the two groups are included. Statistical analysis was performedon dCt values [Ct(target) 2 Ct(reference)].

2828 Diabetes-Protective Diet Across Two Generations Diabetes Volume 63, August 2014

profoundly expressed in the offspring of GF diet–fed micecompared with STD diet–fed offspring, and they are allcharacteristics of immunosuppressive M2 macrophages,whereas the expression of the Nos2 (iNos) gene markerof M1 macrophages was low. However, gene expressionsof intestinal cytokines Il10 and Il17, which have previ-ously been associated with diabetes protection, werelower in these mice similar to Socs1, an inhibitor ofIFN-g signaling and Smad7 and Ski, which are inhibitorsof the regulatory cytokine TGF-b signaling.

Expressions of T-cell marker genes such as Cd3e, Cd8a,and Cd38 were also downregulated in the ileum, includingCcr4 expression, which has been demonstrated on patho-genic autoimmune T cells in NOD mice. Supportive of theflow cytometry results, the CD34 gene expression, whichencodes a key molecule in T-cell trafficking to lymphnodes, was higher in the offspring of GF diet–fed micecompared with STD diet–fed offspring.

Expressions of Intestinal Tight Junction and Mucus-Related Genes Are Elevated in the Gut at WeaningTo investigate whether the more anti-inflammatory geneexpression profile in the gut was associated with animproved intestinal barrier at weaning, gene expressionsof tight junction and mucus components were analyzed inthe ileum and colon. The following tight junctioncomponent genes have been shown to be regulated inassociation with improved intestinal permeability assayresults (29). Gene expressions of Ocln, which encodesoccludin (P , 0.01; Fig. 4A), Tjp1, which encodes tightjunction protein 1 (P , 0.01; Fig. 4B), and Cldn15, whichencodes claudin-15 (P , 0.05; Fig. 4D), were elevated inilea from the offspring of GF diet–fed mice compared withthose of STD diet–fed mice. Ocln (P , 0.05) gene expres-sion was also elevated in the colon together with a tendencyfor higher expression of Muc1, which encodes a proteinthat represents membrane-associated mucin (P = 0.06;Fig. 4E). Conversely, colonic Muc2 expression representingsecreted mucin was lower (P , 0.05; Fig. 4F).

DISCUSSION

The importance of gluten for the development of auto-immune diabetes was previously demonstrated in bothNOD mice and BB rats, and it has become clear thatdisease-modulating interventions, including dietary ormicrobial antigen treatments, in these animal modelsare particularly imperative in early life. To clarify whetherthe effect of gluten could be prevented exclusively bylimiting its exposure in the postnatal period, a two-generation approach was used. Most importantly, thediabetes incidence was significantly reduced in the off-spring of GF diet–fed NOD mice even though they wereweaned to an STD diet. Thus, a GF diet during gestationand lactation was protective later in life. The fact that we,despite the low incidence of T1D in our facility, see a sig-nificant difference in the GF group further substantiatesthe strong effect of this diet. The 30-week-old nondiabeticoffspring of GF diet–fed mice had a lower incidence of

insulitis than the nondiabetic STD mice, which indicatesthat the GF group is not just delayed in diabetes develop-ment, but is as far from diabetic as the nondiabetic con-trol mice that usually do not develop diabetes; however,a longer observational period would be necessary to fullyclarify whether the mice are completely protected againstdiabetes.

In agreement with this result, the ability of a low-protein diet to modify diabetes incidence was reported tocause a significant drop in diabetes incidence from 86% incontrol NOD mice to 63% in NOD mice when given onlyduring pregnancy and lactation (30). In contrast, wheatand barley protein deprivation only until weaning wasdemonstrated not to be sufficient to significantly delaydiabetes development. However, in the gestational andpreweaning period this diet was also supplemented withfish oil and Vit D3, which in the same study (31) weredemonstrated to abrogate the protective effect of a wheatand barley protein-free diet. Interestingly, it was alsoshown that accelerated diabetes was not completelyreconstituted by supplementing the wheat and barley pro-tein-free diet with gliadin, which indicates that other di-etary or microbial antigens also mediate the protectiveeffect. For example, the gut microbiota in GF diet–fedmice, which may not be altered by a pure gliadin supple-ment, is different from that in gluten-fed mice (12). A

Figure 4—Relative gene expression of Ocln (A), Tjp1 (B), Cldn8 (C),Cldn15 (D),Muc1 (E ), andMuc2 (F) in ileum and colon from 4-week-old offspring of GF diet–fed or STD diet–fed mice. Data were nor-malized to Actb and then to the mean control group, which wasdefined as 1. All samples were quantified in duplicate (n = 8–9 pergroup). Error bars represent the SEM. *P < 0.05, **P < 0.01.

diabetes.diabetesjournals.org Hansen and Associates 2829

recent article (17) proposed cereal dietary antigens as astronger T1D inducer than microbes. The authorsreported a similar protective effect on diabetes incidencein BB rats that were fed a low-antigen hydrolyzed casein(HC) diet in both germ-free and specific pathogen–free(SPF) conditions. However, the HC diet was more pro-tective in the germ-free than in the SPF condition, alsoindicating a diabetes-promoting effect of the microbes.This was further supported by a low b-cell mass only inthe cereal-fed BB rats compared with the germ-free andHC-fed BB rats.

In the current study, a distinct bacterial profileenriched in especially Akkermansia, TM7, and Proteobac-teria was evident in both NOD mice fed a GF diet and intheir offspring at weaning. These taxonomic groups werealso previously associated with protection against the de-velopment of autoimmune diabetes in NOD mice onlywhen present before weaning (22), which is interestingas Akkermansia has been demonstrated to modulate hostimmune responses in monocolonized mice (32). In addi-tion, taxonomic differences between the gut microbiomesof healthy and diabetic children were characterized bymucin-degrading Akkermansia, which was more abundantin control subjects than in case patients (33). As glutenhas potential irritating effects in the small intestine whereit is degraded, this might affect mucus production andbarrier function. By leaving out gluten from the diet,the mucus production may increase, which in turn canlead to an increase in the presence and metabolic activityof specific bacterial strains, not least Akkermansia, whichpreviously has been shown to grow on mucus proteins(34). Minor fold-change differences were seen in tightjunction component gene expressions, which indicatedthat Akkermansia might be associated with an improvedintestinal barrier in both the ileum and colon, althoughthis link is purely speculative. The restoration of impairedintestinal barrier and alleviated signs of gut epithelialirritation such as colonic crypt hyperplasia have also pre-viously been shown in response to antidiabetogenic dietsin T1D rodent models (5,35).

Not much is known about the role of TM7 in mam-malian health and disease, but in humans it is mainlyassociated with the oral cavity, where it has been associatedwith periodontitis (36,37). Furthermore, Kuehbacher et al.(38) suggested that TM7 members are involved in theethology of Crohn disease, although the mechanismremains unknown. A detailed examination of the influ-ence of the microbes from GF diet–fed NOD mice bytransferring the microbiota to germ-free mice would beinformative. It is striking that the effect on the microbialcomposition is only present as long as the mice are feda GF diet. This indicates that the effect of the microbiotais dependent on the diet and that this effect is especiallyimportant during the development of the immune sys-tem. Even more striking is the fact that even thoughthe microbiota reverses after the introduction of glutenin the diet at weaning, the mice born by GF mothers are

protected against the development of T1D later in life.Thus, its immune regulatory effect on, for example, Tregsat weaning in the prediabetic stage seems to have a long-lasting impact on the capability of the immune system toprotect against autoimmune attack on the b-cells.

Interestingly, an early article (39) demonstrated thatadoptively transferred T cells from NOD mice on an HCdiet were unable to change the incidence of diabetes andwere presented with similar T-cell receptor–mediated pro-liferative responses compared with controls. Consideringthis, it was hypothesized that immune regulatory mecha-nisms in the pancreatic environment at weaning down-regulate otherwise fully functional diabetogenic T-cellresponse in GF diet–fed NOD mice. In the current study,this hypothesis was supported by increased proportionsof FoxP3+ Tregs in PLN and fewer CD11b+ DCs at wean-ing, which also previously were modulated by an antidia-betogenic diet (5). A similar anti-inflammatory immuneprofile has been observed in BALB/c mice receiving a GFdiet in both pancreatic and gut-associated lymphoid tissue(18). The changes in the immune system were only foundat weaning and not at 10 weeks of age. Thus, it seems thatchanges in immunity later in life when insulitis is moreprogressive are not as critical as in postnatal life for thedevelopment of autoimmune diabetes, during which theinsulitis process begins. This further indicates that the GFdiet, possibly through a change in the gut microbiota, mayhave delayed the development of the adaptive immunity,but that the mice, independently of whether they developT1D or not, eventually develop a mature immune system.

Lower intestinal gene expression of proinflammatorycytokines and higher expression of anti-inflammatory M2macrophage markers, together with increased gut Cd3e,Cd8a expression were found in the offspring of GF diet–fed mice. It is interesting that these findings were alsoseen in SPF BB rats fed an HC diet but not to the sameextent as in the germ-free HC-fed BB rats (17). Thus, itrequired the presence of microbes. M2 macrophages werefurthermore found to be associated with the microbiota-dependent sex difference observed in T1D development inNOD mice (40). The intestinal alterations found in theoffspring of GF diet–fed mice may therefore, also in thisexperiment be a consequence of the altered microbiota inearly life rather than the presence of cereal antigens. Al-though these data cannot be causally linked to the pan-creas, it was demonstrated that an early GF environmentincreased the trafficking of T cells with a mucosal pheno-type to PLN. It would be interesting to further investigatethe regulatory properties of these T cells because a highintestinal IL-15 level, as seen in the GF group, promotesintestinal epithelial cell activation of noncytotoxic CD8T cells with suppressor function (41). These cells havenondetectable granzyme B, which was also expressed sig-nificantly lower in the GF group. This is further consistentwith the prevalence of M2 anti-inflammatory macro-phages as these and Tregs seem to have a mutual abilityto promote the differentiation of one another, at least

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partly through the TGF-b pathway (42,43). It is possiblethat any effect of bacterial or dietary antigens on pancre-atic immune homeostasis would be mediated in part bythe migration of these immune cells that are activated inthe tolerogenic gut environment.

Whether the early ingestion of cereal antigens beforeweaning or the altered microbiota exerts its effect sepa-rately or synergistically is not known. Most importantly,the early GF environment clearly attenuated diabetesdevelopment in the NOD mice even though they wereweaned to a gluten-containing diet, but the changes in gutmicrobiota and immune system were no longer evidentlater in life, from which we can conclude that the time andthe diet before weaning are of imperative importance forprotection against diabetes development.

Funding. This work was carried out as part of the 3G Center—Gut, Grain &Greens; the 3G Center is supported by the Danish Council for Strategic Research.This work was further funded by CHANCE (Chemometric Analysis Centre at theUniversity of Copenhagen), the Center for Applied Laboratory Animal Research,and the Beckett Foundation.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. C.H.F.H. conceived and designed the study, an-alyzed histology, performed flow cytometry, contributed to the discussion, andwrote the manuscript. Ł.K. analyzed the data, contributed to the discussion, andwrote the manuscript. K.B. analyzed histology and contributed to the discussion.S.B.M., C.N., and H.F. were involved in the analysis of gene expression data andcontributed to the discussion. L.H.H. performed pyrosequencing and contributedto the discussion. D.S.N. analyzed the data and contributed to the discussion.S.S. performed flow cytometry and contributed to the discussion. A.K.H. con-ceived and designed the study, contributed to the discussion, and wrote themanuscript. C.H.F.H. is the guarantor of this work and, as such, had full accessto all the data in the study and takes responsibility for the integrity of the data andthe accuracy of the data analysis.

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