The role of the intestinal microbiota in
type 1 diabetes☆
Naoko Haraa, Aimon K. Alkanania, Diana Irb, Charles E. Robertsonc,
Brandie D. Wagnerd, Daniel N. Frankb, e, Danny Ziprisa,⁎
aBarbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, CO 80045, USA
bDivision of Infectious Diseases, University of Colorado School of Medicine, Aurora, CO 80045, USA
cDept. of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, CO 80302, USA
dDepartment of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado Denver,
Aurora, CO 80045, USA
eUniversity of Colorado Microbiome Research Consortium (MiRC), USA
Received 15 November 2012; accepted with revision 1 December 2012
Type 1 diabetes;
Kilham rat virus;
and can influence the balance between pro-inflammatory and regulatory immune responses.
Recent studies suggest that alterations in the composition of the intestinal microbiota may be
linked with the development of type 1 diabetes (T1D). Data from the biobreeding diabetes prone
(BBDP) and the LEW1.WR1 models of T1D support the hypothesis that intestinal bacteria may be
involved in early disease mechanisms. The data indicate that cross-talk between the gut
microbiota and the innate immune system may be involved in islet destruction. Whether a causal
link between intestinal microbiota and T1D exists, the identity of the bacteria and the mech-
anism whereby they promote the disease remain to be examined. A better understanding of the
interplay between microbes and innate immune pathways in early disease stages holds promise
for the design of immune interventions and disease prevention in genetically susceptible
© 2012 Elsevier Inc. All rights reserved.
The digestive tract hosts trillions of bacteria that interact with the immune system
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The intestinal microbiota and the immune system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
☆ This article is dedicated to the memory of Dr. George S. Eisenbarth who passed away on November 13, 2012. Our studies were supported by
grants 1-2006-745, 1-2007-584, 5-2008-224, 5-2011-41, and 17-2011-655 from JDRF.
⁎ Corresponding author at: Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, 1775 Aurora Ct., Mail Stop B-140,
Aurora, CO 80045-6511, USA.
E-mail address: firstname.lastname@example.org (D. Zipris).
1521-6616/$ - see front matter © 2012 Elsevier Inc. All rights reserved.
available at www.sciencedirect.com
Clinical Immunology (2013) 146, 112–119
Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The intestinal microbiome and human T1D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rat models of T1D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The role of the innate immune system in T1D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The intestinal microbiota and T1D in the BBDP rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The role of gut bacteria in virus-induced T1D in the LEW1.WR1 rat . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Type 1 diabetes (T1D) is a proinflammatory disorder that
loss of insulin production . What triggers T1D has not yet
been identified, however evidence from humans and animal
models suggests that in addition to genetics, environmental
factors and microbial infections in particular, may be key
players in disease mechanisms . The incidence of T1D has
substantially increased worldwide in recent decades at a rate
[3,4]. The incidence of T1D has also risen remarkably in very
young children  and T1D can be seen more frequently in
subjects who express low-risk alleles [5,6]. The increase in
disease incidence cannot be accounted for by genetic alter-
ations or improved diagnosis only, and it is hypothesized that
environmental factors play a key role in this phenomenon .
The evidence implicating the environment and microbial
infections in T1D is circumstantial and has been obtained
from epidemiological studies and anecdotal data. For exam-
ple, it was reported that the disease concordance rate of T1D
in monozygotic twins is about 50% . Furthermore, viruses
 and virus-specific antibodies are more commonly found in
individuals with recent disease onset [9–12]. Among viruses
implicated in human T1D are cytomegalovirus [13,14], cox-
sackie B [15,16], mumps , rubella , Epstein–Barr
virus [19,20], rotavirus , and varicella zoster virus .
Reports of an epidemic outbreak of T1D may also imply en-
vironmental factors in the etiology of T1D [23–25]. A sub-
stantial increase in the incidence of T1D in young children
was observed in Philadelphia in the first 6 months of 1993
. Notably, measles epidemic occurred in Philadelphia
approximately two years prior to the outbreak of T1D. This
led to the hypothesis that the increase in the incidence of
T1D could be linked with the measles outbreak . Finally,
 and biobreeding diabetes resistant (BBDR) rat [15,27–29]
and accelerate disease development in the NOD mouse .
Viruses could also prevent disease development in the NOD
mouse if administered to young mice [31,32].
Current studies suggest that the intestinal microbiome
plays a key role in the mechanism(s) of proinflammatory
disorders [33–35]. Alterations in the gut microbial composi-
tion were detected in patients in the early stages of rheuma-
toid arthritis  and inflammatory bowel disease .
Furthermore, spontaneous ankylosing enthesopathy did not
develop in germ-free mice . In contrast, disease was
triggered in animals inoculated with a mixture of anaerobes,
but not with Lactobacillus spp. or Staphylococcus spp. .
Intestinal bacteria were recently linked with T1D in humans
, the BBDP rat , the LEW1.WR1 rat model of virus-
induced T1D , and the non-obese diabetic (NOD) mouse
[42,43]. How the gut microbiota is involved in mechanisms
leading to T1D is not yet clear. We have recently addressed
the hypothesis that interactions between the intestinal
microbiota and the innate immune system are associated
with the course of T1D in the LEW1.WR1 rat model of T1D.
Here, we will discuss findings from our recent studies ad-
dressing the role of the gut microbiota-innate immunity axis
in the development of T1D in rat models of T1D. Data from
the NOD mouse will not be discussed, as excellent reviews
discussing this topic in this animal model have recently been
published [For example, see ref. ].
2. The intestinal microbiota and the
Approximately 100 trillion bacteria reside on or in the human
body most of which colonize the intestine . Consequently,
the gastrointestinal tract is the primary site of interactions
between microbes and the host immune system [reviewed
in refs. [35,46–48]]. 16S rRNA-based approaches demonstrat-
ed that members of the bacterial phyla Bacteroidetes and
Firmicutes constitute more than 90% of the known phyloge-
netic diversity within the distal gut microbiota . A high
degree of diversity of the gut microbiome exists between
gut microbiome becomes less diverse later in life . The
intestinal bacteria play an essential role in gut development,
nutrition, as well as the development of fully functional
immune system [34,45,51,52]. For example, germ-free ani-
mals that lack intestinal bacteria display extensive abnormal-
ities in the development of gut-associated lymphoid tissues
[53,54] and in antibody production, have fewer and smaller
Peyer's patches and mesenteric lymph nodes. Furthermore,
the CD4+CD25+Treg cells of these germ-free animals express
lower levels of FoxP3 and are functionally abnormal as com-
pared with animals housed under specific pathogen free
environment . Changes in the composition of the gut
microbiome also are associated with mechanisms of intestinal
autoimmunity and obesity . Altered development or com-
position of the gut microbiota is likely to disturb the cross-talk
between the microbiota and the host immune system and
thereby contribute tochronic inflammationas issuggested for
inflammatory bowel disease [34,37,55–58]. To keep the in-
testinal microbiota in check and sustain intestinal homeosta-
sis, the host must maintain immune mechanisms that can
control bacterial growth and composition. Epithelial cells are
113The role of the intestinal microbiota in type 1 diabetes
[59,60]]. They express pattern recognition receptors (PRRs)
such as Toll-like receptors (TLRs) and Nod-like receptors
(NLRs) that interact with molecular structures expressed by
bacteria termed microbial-associated molecular patterns
(MAMPs). Interactions between PRRs and MAMPs lead to the
upregulation of anti-bacterial molecules and chemokines that
can in turn recruit immune cells to initiate adaptive immune
responses . In addition to stimulating innate immunity,
bacterial colonization of the intestines induces adaptive
immune responses, such as the expression of secretory IgA,
the differentiation of effector Th1, Th2, and Th17 cells, as
well as the development of Treg cells .
3. The intestinal microbiome and human T1D
Very little is presently known about the role of the intestinal
microbiota in the development of human T1D. Results from
the Diabetes Prediction and Prevention study (DIPP) per-
formed in Finland indicated that children who progressed to
T1D had reduced relative abundances of Firmicutes and
increased Bacteroidetes over time, whereas age- and HLA-
matched healthy children had relatively increased Firmicutes
and reduced Bacteroidetes . Interestingly, all the differ-
within the Bacteroidetes phylum and the bacterial genus
Bacteroides; more than 20% of the observed shift was at-
tributed to the species Bacteroides ovatus. Most of the
increase in the abundance of Firmicutes detected in healthy
subjects was a result of increased abundance of the order
Clostridiales, more specifically the bacterial strain CO19.
Healthy children had a more diverse and stable intestinal
autoimmunity. The size of the study sample was too small to
arrive at strong conclusions, however it was proposed that
that an “autoimmune microbiome” may be involved in pre-
disposing genetically susceptible individuals to T1D .
Studies are currently underway to identify the gut bacterial
composition in the intestine from a large sample of at risk
subjects enrolled in The Environmental Determinants of
that the data from this study will shed new light on the role of
intestinal microorganisms and other potential environmental
triggers in the development of T1D.
4. Rat models of T1D
The diabetes-prone biobreeding (BBDP) rat develops sponta-
neousdiabetes, whereasthediabetes-resistant BB(BBDR)rat
is diabetes-free in a specific pathogen free environment
[61,62]. The BBDP is severely deficient of T lymphocytes due
to a mutation in the gene encoding the mitochondrial mem-
brane protein Ian4, a defect that results in a decrease in
the peripheral T cell life span . BBDR rats were derived
from BBDP forebears by selection for the absence of disease
. Both BBDP and BBDR rats express RT1uMHC haplotype
[62,65]. In contrast to the BBDP rat, the BBDR model has
normal levels and function of peripheral CD4+and CD8+T
cells [62,65] and spontaneous T1D does not occur in this
animal . The BBDP rat develops spontaneous T1D with
characteristics similar to those of the human disease [61,62].
The LEW1.WR1 rat has normal T cell proportions and T cell
function . Type 1 diabetes can be triggered in ~50% of
animals infected with the parvovirus Kilham rat virus (KRV)
[68,69].Thedisease isimmune mediated,asisletdestruction
can be ameliorated with therapies directed against theT-cell
receptor, CD5, or CD8 [66,70]. Furthermore, transferring
spleen cells from virus-infected rats to class IIucompatible
rats transfers insulitis and T1D [66,70]. T1D induced by KRV
can be observed beginning on day 14 following virus inoc-
ulation, and the disease is characterized by specific loss
of islet beta cells, glycosuria, ketonuria, and polyuria in a
strain-specific manner [64,66,67,70,71]. Susceptibility to
virus-induced disease is dependent on the presence of class I
Auand class II B/Du[66,70]. Other than virus infection,
LEW1.WR1 rats can develop T1D following the depletion of
ART2.1 expressing CD4Tcells .How infection with a virus
leads to T1D is not fully understood. Direct killing of insulin
producing cells by KRV is unlikely to be part of disease
mechanisms, since similar expression levels of insulin are
detected in islets from infected versus uninfected animals
on day 5 following infection at the time when transcripts for
KRV are detected in islet beta cells (our unpublished data).
Rather, we propose that KRV infects pancreatic lymph nodes
and islet beta cells and induces a proinflammatory response
culminating in the recruitment of innate and adaptive im-
mune cells to the site of inflammation and ultimately islet
5. The role of the innate immune system in T1D
Earlier studies have highlighted the complex role that the
innate immune system may play in the course of T1D. TLR-
induced innate immune signaling plays a crucial role in
virus-induced T1D in the LEW1.WR1 and the BBDR models
[2,26,68,69]. KRV activates plasmacytoid DCs and B cells to
produce pro-inflammatory cytokines and chemokines in vitro
and in vivo via a mechanism linked with TLR9 pathways
[28,29]. Treatment with chloroquine, a TLR9 antagonist used
in the clinic to treat malaria, or steroidal and non-steroidal
anti-inflammatory agents can suppress KRV-induced in vitro
proinflammatory cytokine production and prevent islet
destruction in vivo [our unpublished data and refs. [29,71]].
Virus infection combined with a brief pretreatment with
polyinosinic:polycytidylic acid (poly I:C), a synthetic analog
of double stranded RNA and an agonist of TLR3 and MDA5,
induces islet destruction in the majority of the treated
animals, implying that innate immune activation combined
with virus infection can exacerbate islet autoimmunity .
KRV infection induces the expression of transcripts for the
virus and the upregulation of proinflammatory molecules in
the pancreatic lymph nodes and islet beta cells 5 days fol-
lowing virus infection [our unpublished data and ref. ].
Treatment with Poly (I:C) alone at doses that combined with
KRV enhance T1D does not lead to islet destruction, em-
phasizing the importance of live virus infection in disease
induction . How Poly (I:C) enhances virus-induced T1D is
unknown. It is clear, however, that the mechanism of disease
exacerbation does not involve enhancement of inflammation
early after infection . In the BBDP rat, administering Poly
(I:C) at relatively high doses can accelerate T1D . Adding
to the complexity, injecting these animals with Poly (I:C) at
114 N. Hara et al.
doses that are 100 times lower than those required for disease
acceleration can prevent T1D presumably via interfering with
the development of insulitis . Finally, treating NOD mice
with Poly (I:C) completely blocks islet destruction . How
the interplay between the innate immune system and
adaptive immunity results in islet autoimmunity or immune
tolerance remains to be determined.
6. The intestinal microbiota and T1D in
the BBDP rat
The hypothesis that the intestinal microbiome is involved in
the development of systemic autoimmune disorders has re-
cently gained momentum. Studies performed in humans with
autoimmunity support this possibility; however these investi-
gations could not establish a direct link between altered
intestinal microbiota and disease development. The role of
the gut microbiota in mechanisms of autoimmunity can be
more directly addressed using animal models. Current data
indicate that alterations in gut bacteria are detectable prior
totheonsetof T1DinboththeBBDPratand theLEW1.WR1rat
models of T1D, raising the possibility that these changes could
be linked with early disease mechanisms. The first evidence
implicating the intestinal microbiota in the course of T1D in
the rat was provided by Brugman and colleagues [Table 1,
Fig. 1, and ref. ]. They documented that BBDP rats that
progressed to T1D had lower abundances of Bacteroides as
compared with rats that remained disease-free. Antibiotic
therapy or antibiotic treatment combined with hydrolyzed
casein diet ameliorated T1D via mechanisms associated with
changes in gut bacterial communities . Furthermore,
diabetes-prone BBDP rats had reduced abundances of the
bacterial genera Lactobacillus and Bifidobacterium as com-
bacterial composition that predisposes to T1D .
Why BBDP rats have altered gut microbiome is currently
unclear. However, because this animal has severe lympho-
penia , and since the immune system can greatly in-
fluence the gut bacterial composition , it is plausible
that the changes in the microbiome could be associated
with the abnormal immune system intrinsic to this model.
Whether the altered intestinal microbiome is causally linked
with disease occurrence in the BBDP model remains to be
addressed . Interestingly, the transfer of Lactobacillus
johnsonii N6.2 isolated from the BBDR intestine to BBDP rats
Table 1The effect of manipulating the intestinal microbiome on T1D in the rat.
Treatment Effect on
Potential mechanisms involved
in disease manipulation
BBDP Spontaneous Oral SulfatrimPrevention Reduction in the abundance of
Altered gut bacteria (?)
Oral Sulfatrim plus hydrolyzed
Oral administration of
Lactobacillus johnsonii strain
N6.2 isolated from the intestine
of the BBDR rat model
Upregulation of Th17 cells in
mesenteric lymph nodes
LEW1.WR1KRV Prevention Reversal of KRV-induced alterations
in the gut microbiota;
Downmodulation of inflammation in
pancreatic lymph nodes and Peyer's
Downregulation of adaptive immunity
in the spleen
treatment group in LEW1.WR1. The relative percent abundances
ofintestinal bacterial phyla are inferredfrom 16S rRNA sequence
counts in datasets. Bacterial profiles from rat fecal DNA samples
were determined by broad-range PCR of the 16s V1V3 variable
region andphylogeneticsequence analysis. Amplicons ofthe
16S rRNA gene (~500 base pairs; primers 27FYM+3 and 534R)
[84,85] were generated via broad-range PCR (26 cycles) using
5′-barcoded reverse primers . PCR yields were normalized
using a SequalPrep™ kit (Invitrogen, Carlsbad, CA), pooled,
lyophilized, and purified using a DNA Clean and Concentrator Kit
(Zymo, Irvine, CA) . Pooled amplicons were pyrosequenced
on a GS Junior 454 sequencing instrument using Titanium
chemistry (Roche Life Sciences, Indianapolis, IN). The sequences
werefurther analyzed as describedin ourrecent report .The
X axis represents percentages of bacterial phyla. The Y axis
represents the treatment groups. CTRL, samples from uninfected
with virus plus Sulfatrim; S, animals treated with Sulfatrim only.
Operational Taxonomic Units (OTU) of phyla from
115 The role of the intestinal microbiota in type 1 diabetes
delayed disease development in a bacteria-specific manner
 via a mechanism that may involve the upregulation of
Th17 cells . These findings are reminiscent of recent
data from the NOD mouse demonstrating that natural trans-
mission of segmented filamentous bacteria to this mouse
correlates with disease prevention and the upregulation of
Th17 cells in the intestine . It suggests a common mech-
anism of bacteria-induced disease amelioration in the BBDP
and the NOD model systems. Taken together, the data imply
that bacteriotherapy may potentially be used as a means to
modulate the immune system and interfere with the course
of autoimmunity for preventing islet destruction [Table 1
and ref. ]. To what extent the protective bacteria in fact
modulate the gut microbiome remains to be seen.
7. The role of gut bacteria in virus-induced T1D
in the LEW1.WR1 rat
Does KRV infection induce alterations in the gut bacterial
composition in the LEW1.WR1 model and are intestinal bac-
teria involved in the course of virus-induced T1D? Experi-
ments addressing this possibility revealed that KRV induces a
transient alteration of the gut microbiota represented by
an increase in the abundance of the Actinobacteria phylum
and the Bifidobacterium genus on day 5 but not day 12 post-
infection [Table 1, Fig. 1, and ref. ]. A transient increase
in the abundance of Clostridium and changes in the ratio
between bacterial communities shortly after infection
provided further support to the hypothesis that intestinal
bacteria may be involved in the course of islet destruction in
the LEW1.WR1 model [Table 1, Figs. 1, 2, and ref. ]. How
KRV triggers changes in gut bacteria is currently unknown.
We favor the hypothesis that these alterations are a con-
sequence of the proinflammatory state induced by KRV in
Peyer's patches and other lymphoid organs shortly after
infection . Experiments are underway to determine
whether changes in the gut microbiota in virus infected
LEW1.WR1 rats are directly linked with disease mechanisms.
Further support to the possibility that the gut microbiome
is involved in the course of virus-induced disease was provided
by the observation that therapy with the broad spectrum
antibiotic Sulfatrim can prevent insulitis and islet destruction
via a mechanism that could involve downmodulation of KRV-
induced innate and adaptive immune responses . For
example, Sulfatrim reduced the level of transcripts for IRF-7,
CXCL-10, IL-17A, and IL-6 in pancreatic lymph nodes and
Peyer's patches shortly after infection . Consistent with
these findings, Sulfatrim downregulated B cells previously
documented to be involved in virus-induced inflammation
 as well as lowered the proportion of anti-KRV-specific T
cells in the spleen. In any case, the protective effect of
Sulfatrim is unlikely tobelinkedwithchanges inviral loads, or
pancreatic lymph nodes .
The observations indicating that gut bacteria can impact
innate and adaptive immunity beyond the gut and alter
the course of autoimmunity in animal models are compatible
with earlier data from other diseases and may have an
important clinical implication [reviewed in ref. ] as they
point to the possibility that targeting the gut microbiome
prior to disease onset may potentially be used as a disease
intervention approach. Similar to the LEW1.WR1 rat, antibi-
otic therapy can regulate distal immune responses in the
respiratory mucosa . Along this line, mice deficient in
GPR43, a receptor that recognizes acetate and propionate,
the products of fiber metabolism by intestinal bacteria, have
exacerbated inflammation in the KxB/N arthritis model and
in a model of allergic airway inflammation .
The data implying that intestinal bacteria are involved in
promotingdisease inthe LEW1.WR1 modelare consistentwith
the overall notion that microbial infections and TLR-induced
innate activation promote islet autoimmunity in the rat .
The KRV-induced altered intestinal microbiota in LEW1.WR1
rats does not involve major differences in the composition of
the gut bacterial composition in this animal, as it was found to
be similar to that found in mice and humans and composed of
few other less abundant bacterial types. One possibility that
gut microbiome lead to altered gut permeability and conse-
quently to the passage of microbes beyond the gut epithelial
with KRV. DNA was extracted from fecal samples collected from individual rats on day 5 following viral inoculation. Quantitative PCR
analysis was used to assess the abundance of bacterial communities, as indicated in the figure. Standard curves were obtained using
DNA extracted from a fecal sample from a naïve C57BL6 mouse. DNA levels were calculated using a bacterial reference gene. The
calculated ratios between the expression level of Bifidobacterium relative to Lactobacillus, Clostridium, or Bacteroides are shown as
indicated in the figure. Bars represent mean values. Statistical analyses were performed using the non-parametric Mann–Whitney
Ratios between gut bacterial communities in KRV-infected LEW1.WR1 rats. Animals were either untreated, or injected
116 N. Hara et al.
barrier culminatingintheupregulation of anti-isletTcellsand
islet destruction T1D .
Emerging evidence indicates that the intestinal microbiota
promotes the development of the immune system and that
performed in humans and animal models implicate the in-
testinal microbiota in the development of islet autoimmunity.
However, a number of major questions in this area remain to
be resolved. A key issue is what are the bacterial communities
involvedintriggeringor preventingT1D andhow they, ortheir
metabolites, alter the balance between proinflammatory and
regulatoryimmuneresponses inpancreatic islets.Otherissues
that await further examination are related to the timing with
which thealtered gutmicrobiome occursinrelationtodisease
development and the trigger(s) leading to these changes.
Addressing these issues is essential for a better understanding
of early mechanisms of the disease in genetically-susceptible
individuals. This could lead to the development of new ap-
proaches to modulate the immune system and prevent T1D.
Conflict of interest statement
The author(s) declare that there are no conflicts of interest.
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