Naturally transmitted segmented filamentous bacteria
segregate with diabetes protection in nonobese
Martin A. Kriegela,b, Esen Sefika, Jonathan A. Hilla,1, Hsin-Jung Wua,2, Christophe Benoista,3, and Diane Mathisa,3
aDepartment of Pathology, Harvard Medical School, Boston, MA 02115; andbDivision of Rheumatology, Immunology, and Allergy, Brigham and Women’s
Hospital, Boston, MA 02115
Contributed by Diane Mathis, June 7, 2011 (sent for review May 18, 2011)
Vertebrates typically harbor a rich gastrointestinal microbiota,
which has coevolved with the host over millennia and is essential
for several host physiological functions, in particular maturation of
the immune system. Recent studies have highlighted the impor-
tance of a single bacterial species, segmented filamentous bacteria
(SFB), in inducing a robust T-helper cell type 17 (Th17) population
in the small-intestinal lamina propria (SI-LP) of the mouse gut.
Consequently, SFB can promote IL-17–dependent immune and au-
toimmune responses, gut-associated as well as systemic, including
inflammatory arthritis and experimental autoimmune encephalo-
myelitis. Here, we exploit the incomplete penetrance of SFB colo-
nization of NOD mice in our animal facility to explore its impact on
the incidence and course of type 1 diabetes in this prototypical,
spontaneous model. There was a strong cosegregation of SFB
positivity and diabetes protection in females, but not in males,
which remained relatively disease-free regardless of the SFB sta-
tus. In contrast, insulitis did not depend on SFB colonization. SFB-
positive, but not SFB-negative, females had a substantial popula-
tion of Th17 cells in the SI-LP, which was the only significant,
repeatable difference in the examined T-cell compartments of
the gut, pancreas, or systemic lymphoid tissues. Th17-signature
transcripts dominated the very limited SFB-induced molecular
changes detected in SI-LP CD4+T cells. Thus, a single bacterium,
and the gut immune system alterations associated with it, can
either promote or protect from autoimmunity in predisposed
mouse models, probably reflecting their variable dependence on
different Th subsets.
gender|microbiome|T lymphocyte|autoimmune disease
brates by a multitude of microbes is evolutionarily conserved
and is essential for many of the host’s physiologic processes, in-
cluding proper maturation of the immune system (1–3). Indeed,
animals housed under germ-free (GF) conditions have several
dysfunctional or deficient immunological compartments, result-
ing in abnormal responses to a variety of immune challenges (4).
Recent advances in the molecular discrimination of com-
mensal bacteria colonizing the gastrointestinal tract of mice and
in the flow cytometric parsing of murine gut lymphoid com-
partments have uncovered specific effects of certain bacterial
species or genera on particular lymphocyte subsets. For example,
the gut-resident commensal, segmented filamentous bacteria
(SFB), promotes the development of a robust T-helper cell type
17 (Th17) population in the small-intestinal lamina propria (SI-
LP) (5, 6), whereas Clostridia species induce forkhead box P3-
positive (Foxp3+) regulatory T cells (Treg) in the colonic lamina
propria (7). Relatedly, colonization of the mouse gastrointestinal
tract with the human gut commensal Bacteroides fragilis or ad-
ministration of one of its products, polysaccharide A, can have
a substantial impact on both effector and regulatory T-cell sub-
sets, depending on the context (8–10). In addition, bacterial
DNA or bacterially produced metabolites, such as ATP or short-
olonization of the external and mucosal surfaces of verte-
chain fatty acids, can exert profound effects on both the innate
and adaptive gut immune systems (11–13).
Not unexpectedly, then, gastrointestinal microbiota can alter
the incidence and severity of gut autoimmune/inflammatory
diseases (14, 15); in fact, inflammatory bowel disease is “trans-
missible” via gut-resident microbiota under certain experimental
conditions (16). More surprising have been recent observations
that SFB’s influence can be translated systemically to trigger
autoimmune arthritis in a predisposed mouse strain (17) or to
exacerbate experimental encephalomyelitis (18). Both these
diseases depend critically on Th17 cells, a likely explanation for
the SFB influence.
Type 1 diabetes (T1D)—especially in the prototypical, sponta-
neous NOD mouse model (19) —provides an interesting coun-
terpoint. The incidence of T1D is higher in countries with stricter
hygiene practices (reviewed in refs. 20, 21), an observation paral-
leled by the finding that the diabetes incidence in NOD mice often
is higher in cleaner colonies, being fully penetrant in GF con-
ditions (22, 23). Furthermore, a null mutation of MyD88, an in-
nate immune system signaling molecule, altered the composition
of the gut microbiome and prevented disease development in
NOD mice housed in a specific-pathogen-free (SPF) facility (24).
The protective effect was attributed to commensals otherwise kept
in check by MyD88 signaling, because mice expressing or not
expressing this molecule were equally susceptible to disease when
housed GF (24). However, the specific commensals responsible for
protection from diabetes under routine husbandry conditions re-
main to be identified.
Here, we have exploited the particular make-up of our ex-
perimental NOD mouse colony to perform a segregation analysis
of diabetes development relative to SFB colonization. Cose-
gregation of SFB positivity and protection from diabetes was
subtended by the appearance of a robust Th17 compartment in
the SI-LP as well as an increased representation of Th17 signa-
ture transcripts, specifically in SI-LP CD4+T lymphocytes.
Facility and Individual Variation in Colonization of the Gastro-
intestinal Tract of NOD Mice by SFB. It was reported more than 3
decades ago that microscopically defined SFB can variably col-
Author contributions: M.A.K., E.S., J.A.H., H.-J.W., C.B., and D.M. designed research; M.A.K.,
E.S., J.A.H., and H.-J.W. performed research; M.A.K., E.S., J.A.H., H.-J.W., C.B., and D.M.
analyzed data; and M.A.K., C.B., and D.M. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The datasets reported in this paper are available at the National Center
for Biotechnology Information (accession no. 29806).
1Present address: Tempero Pharmaceuticals, Inc., Cambridge, MA 02139.
2Present address: Department of Immunobiology, University of Arizona, Tucson,
3To whom correspondence may be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1108924108PNAS Early Edition
| 1 of 6
onize the same mouse strain in different animal facilities (25).
More recently, Littman and colleagues, applying a more de-
finitive 16S rRNA quantification method, demonstrated that
SFB was present in C57BL/6 (B6) mice housed at Taconic Farms
(TAC) but was absent from individuals of the same strain kept at
the Jackson Laboratory (JAX) (6). However, it was unclear to
what extent all individuals of all strains housed at a particular
facility shared SFB status, whether positive or negative. Our
primary focus was SFB colonization of the NOD strain, in par-
ticular within our experimental colony at the New Research
Building (NRB) of Harvard Medical School.
Relative SFB levels in fecal pellets of mice housed at the three
sites were determined by quantitative PCR assays that used both
specific SFB and conserved eubacterial (EUB) 16S rRNA pri-
mers, the latter capable of amplifying the RNA for most bacte-
rial species to permit normalization. [We previously had
established that relative SFB levels estimated from fecal pellets
reflected well those in the distal small intestine (Fig. S1)]. As
anticipated, SFB colonization varied at the different animal fa-
cilities, the “cleanest” site being JAX, which showed undetect-
able levels in the three strains examined (Fig. 1A). Less expected
was that TAC harbored both SFB-positive (B6, BALB/c) and
SFB-negative (NOD) strains. Interestingly, BALB/c and NOD
mice from the NRB facility were variably colonized with SFB;
this variation probably reflects the fact that this colony is
replenished periodically with new mice from an SFB-negative
seed colony that we maintain in Bar Harbor.
Fig. 1B shows the pattern of SFB transmission within a group of
NOD mice housed at the NRB facility, followed across four
generations (I–IV) in 2010. Various modes of bacterial transfer
are in evidence. (i) Vertical passage by the mother is illustrated by
breeding cage J. All offspring from this cage born on either 10/23
or 11/29 were SFB-positive, indicating complete maternal trans-
mission. (ii) Horizontal passage is suggested by breeding cage I.
The father in this cage derived from SFB-negative parents and
was a littermate of the six SFB-negative offspring of cage D.
Likely, he was colonized through cohousing with the unrelated
SFB-positive female with which he mated in cage I. Indeed, in-
dependent cohousing experiments with mice of the same sex but
opposite SFB status have confirmed horizontal transmission
within 1–2 wk of cohousing. (iii) Incomplete vertical passage is
illustrated by the mixed SFB status of the offspring of breeding
cage A, suggesting that this bacterium may be lost over time,
leading to incomplete transmission for longer-term cages, result-
ing in SFB-negative offspring until an SFB-positive breeder is
again introduced into the line (as per breeder cage I).
In short, different animal facilities, different strains within the
same facility, or different individuals within the same strain can
show divergent SFB colonization. Given the important impact of
SFB on the immune system (5, 6, 17, 26), this variable needs to
be taken into account more seriously when considering data from
Protection from Type 1 Diabetes in Female NOD Mice Segregates with
SFB Colonization. Diabetes incidence in NOD mice housed at the
NRB facility is about 50–60% for females and 10–15% for males,
Relative SFB Level (Feces)
N.D. N.D. N.D.
SFB levels in mice housed at TAC, JAX, or the NRB facility. Each bar represents the average level +/− SEM for six mice (three males and three females). Fecal
pellets taken from mice that were shipped to the NRB facility from TAC or JAX were collected and processed or were frozen within 24 h of arrival. Mice were
5–6 wk of age at the time of feces collection. SFB negativity in the NOD strain was confirmed 10 months later with additional sets of mice at 6–8 wk of age. (B)
SFB colonization through four generations of NOD mice housed at the NRB facility. Squares represent males; circles indicate females. Black symbols indicate
SFB-positive mice; white symbols indicate SFB-negative mice; mixed black/white symbols indicate mice of unknown SFB status. Each litter is delineated by the
brackets below, with the date of birth (month/day) within 2010 indicated. SFB status of pups was assessed by PCR of fecal DNA at 4–6 wk of age except for
breeding cages A–J. Parental feces were collected and examined for the presence of SFB within 2–3 wk of the litter encompassing the line of descent. The
parental age at the time of feces collection varied between 2.5 and 6 mo.
SFB status of mice housed in different animal facilities. (A) Variable SFB colonization of different strains at the different sites. PCR-determined relative
P < 0.0001
P = ns
#1#2#3 #4#1#2 #3#5#4
positive NOD mice at the NRB facility. (A and B) Cumulative incidence curves
for female (A) and male (B) NOD mice identified as SFB positive or SFB
negative at 4–6 wk of age. Diabetes development was monitored weekly
starting at 10 wk of age. The difference between the two groups was sta-
tistically significant in females (P < 0.0001, log-rank test) but not in males. (C)
Insulitis scores from SFB-colonized female NOD mice at 10 wk of age and
age-matched SFB-negative controls. Pancreata were prepared and stained
with H&E, and islet cell infiltration was scored as described in SI Materials
and Methods. Each bar represents an individual mouse.
Diabetes incidence and insulitis scores in SFB-negative and SFB-
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in the lower half of the world-wide range (22) and below the
∼80% and 90% incidence currently reported on the websites of
the TAC and JAX facilities, respectively. Wondering whether
the lower penetrance at our facility might somehow reflect par-
tial colonization by SFB, we cotracked disease development and
SFB status in 41 SFB-negative and 42 SFB-positive NRB-housed
NOD mice. There was a clear-cut segregation of high diabetes
incidence with SFB negativity in the females: only 16% of those
colonized by SFB developed disease by 30 wk of age, but 91% of
those that were SFB negative did (Fig. 2A). The latter value is
similar to the high incidences of diabetes mentioned above for
the SFB-negative NOD colonies at TAC and JAX. In contrast,
there was no such correlation in males housed at the NRB fa-
cility; the incidence of diabetes was similarly low, <20%, whether
or not they were colonized with SFB (Fig. 2B).
Insulitis was similar in level and appearance in 10-wk-old SFB-
negative and SFB-positive females, most individuals of both
types showing at least some degree of frank islet infiltration (Fig.
2C). This finding argues that SFB colonization does not block
the disease trigger in NOD mice but instead somehow may
modulate diabetes unfolding.
Induction of IL-17–Producing CD4+T Cells in the SI-LP of SFB-Positive
NOD Females. Given reports that SFB rather specifically promotes
the development of a robust Th17 population in the SI-LP of B6
mice (6, 17), we used multiparameter flow cytometry to compare
the SI-LP and related lymphoid compartments of SFB-positive and
SFB-negative individuals of the NOD strain. Indeed, there was
a clear induction of IL-17–expressing CD4+TCRβ+cells in the SI-
LP of NOD females colonized with SFB vis-à-vis those not colo-
nized (Fig. 3A). The fraction of IL-17–expressing cells varied widely
in individual SFB-positive females (Fig. 3B), but there was a good
correlation between this fraction and the fecal pellet SFB titer (Fig.
3C). In contrast, there was no significant induction of Th17 cells
in the colonic lamina propria, spleen, or pancreatic lymph nodes
(PLN), the site where naive diabetogenic T cells are primed initially
by pancreas-derived antigens (27) (Fig. 3 A and B). Nor was there
a significant enrichment of the IFN-γ–expressing Th1 population in
any of the lymphoid tissues examined (Fig. 3 A and D).
Because it has been argued that Foxp3+CD4+Treg cells may
have a reciprocal relationship with Th17 cells (28, 29), we paid
special attention to this subset. However, SFB-positive and SFB-
negative NOD females housed at the NRB facility had very
similar Treg fractions in each of the four lymphoid tissues ex-
amined, notably in the colonic lamina propria (Fig. 3 E and F).
More broadly, we saw no substantial, repeatable differences in
the total αβ, total CD4+, CD8+, or γδ T-cell compartments of
SFB-colonized and SFB-free female NOD mice. For all of the
populations examined to date, males and females were indis-
tinguishable; most importantly, SFB-negative NOD males, just like
their female counterparts, had few IL-17–producing CD4+TCRβ+
cells in the SI-LP and colon lamina propria, and, vice versa, the
SFB-positive male and female pairs examined concomitantly had
similarly high numbers of SI-LP Th17 cells (Fig. 4).
Specific Induction of Th17 Signature Genes in CD4+T Cells from the
SI-LP of SFB-Positive NOD Mice. To explore further SFB-induced
changes in the CD4+T-cell compartment of the SI-LP in an un-
biased manner, we performed gene-expression profiling using
Affymetrix microarrays. The profiles of CD4+SI-LP T cells from
6- to 10-wk-old SFB-positive (n = 4) and SFB-negative (n = 6)
NOD mice were quite similar overall: In individuals colonized by
SFB, only 55 and 19 transcripts were up- and down-regulated,
respectively, at an arbitrary fold-change threshold of 2 [see
SI-LP %IL-17 expressing
CD4+ TCR-β+ cells
Relative SFB Level (Feces)
R = 0.758
P = 0.015
CD4+ TCR-β+ cells
CD4+ TCR-β+ cells
P = 0.022
SpleenSI-LP Colon-LP PLN
CD4+ TCRβ+ cells
or SFB-negative female NOD mice at 6–8 wk of age. Intracellular cytokine expression was enhanced by a 4-h culture in the presence of phorbol ester and
ionomycin. Cells were gated by side-scatter and as CD45+, CD19−, CD8−, TCRβ+, and CD4+. Shown is a representative plot for IL-17 and IFN-γ staining from six
mice analyzed. (B) Frequencies of IL-17–producing CD4+TCRβ+cells in various lymphoid organs of SFB-positive and SFB-negative NOD mice. Gating was done as
per A. Statistical analysis was performed using the nonparametric Mann–Whitney test. (C) Correlation of relative SFB levels in fecal pellets (determined as per
Fig. 1) and percentage of IL-17–expressing CD4+TCRβ+cells (as per A and B). Statistical analysis was performed using the Pearson correlation. (D) Frequencies
of IFN-γ–producing CD4+TCRβ+cells in the same lymphoid organs of the NOD mice depicted in B. Gating was done as per A. (E and F) Representative flow
cytometry plots of three mice analyzed and frequencies of Foxp3-expressing CD4+TCRβ+cells for various lymphoid tissues of age-matched SFB-positive and
SFB-negative NOD mice. Cells were gated by side-scatter and as CD45+, CD19−, CD8−, TCRβ+, and CD4+. Ns, not significant.
Induction of Th17 cells in the SI-LP of SFB-positive NOD females. (A) Lymphocytes were isolated from the indicated lymphoid tissues of SFB-positive
Kriegel et al.PNAS Early Edition
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Fig. 5A, and Table S1 for raw data and false discovery rates
(FDR) and Fig. S2 for individual gene plots], compared with 16
and 15 transcripts over- or underrepresented, respectively, in
randomized control datasets. Consistently up-regulated in SFB-
positive NOD mice was a set of Th17 signature genes, including
Il17a,Il17f, Il22,Il1r1, andIl23r (indicated in red). In contrast, the
levels of transcripts characteristic of Th1, Th2, and Treg cells
(shown in blue) were not influenced by SFB status. SFB coloni-
zation also increased the representation of Il2 and Il21 transcripts
in CD4+SI-LP T cells, of interest because the corresponding
genes fall within one of the more potent diabetes susceptibility
intervals, idd3 (30). In line with the flow cytometric data (Fig. 3 A
and B), Th17 signature genes were not induced in CD4+T cells
residing in the colonic lamina propria (Fig. 5B and Table S2).
The heatmap of Fig. 5C reemphasizes the specific induction of
transcripts characteristic of Th17 cells in the CD4+SI-LP T cells
of SFB-positive versus SFB-negative NOD mice housed at the
NRB facility. It also illustrates the striking difference vis-à-vis
splenic CD4+T cells, which show no signs of induction, again
consistent with the flow cytometry data (Fig. 3 A, B, and D).
That autoimmune diabetes is exacerbated in NOD mice housed
under GF conditions was first reported almost 25 y ago (23),
prompting widespread speculation that commensal microbiota
normally keep islet-directed autoimmunity in check. An influence
of gut-resident microbes would be consistent with several indi-
cations of a privileged link between the gut and pancreas/PLN,
notably a preferential antigen-trafficking route (31) and shared
lymphocyte homing addressins (32, 33). However, more recent
reports that GF and SPF NOD mice show very similar disease
profiles (24, 34, 35) have clouded the issue. This report offers an
explanation for at least some of these discrepancies. Extending
recent observations that B6 mice housed in the TAC and JAX
animal facilities were differentially colonized by SFB (6), we
showed that NOD mice kept at different sites also carried dif-
ferent loads of this gut commensal. In our variably infected NRB
colony, SFB-negative females had a high diabetes incidence,
approaching complete penetrance, whereas SFB-positive females
were protected from diabetes. Obviously, then, disease exacer-
bation when comparing GF and SPF mice would be most evident
in a colony infected with SFB and would be only minimal in an
CD4+ TCR-β+ cells
NOD mice classed by SFB status. Frequencies of IL-17–producing CD4+TCRβ+
cells in various lymphoid organs of SFB-positive and SFB-negative female and
male NOD mice. Lymphocytes were isolated and stained and were gated as
per Fig. 3A. IL-17 expression levels from four SFB-positive and four SFB-
negative female NOD mice from Fig. 3 were paired with SFB-positive and SFB-
negative male mice processed on the same day. Statistical analysis was per-
formed using the paired t test. No statistically significant differences between
SFB-positive and SFB-negative NOD mice were found in any compartment
except the SI-LP. The P values for SI-LP IL-17 levels from SFB-positive versus
SFB-negative NOD mice are 0.0097 for females and 0.0459 for males.
Comparison of Th17 cell numbers in the SI-LP of female versus male
positive NOD mice. (A) Affymetrix microarray analysis of SI-LP CD4+T cells
from SFB-negative (x axis) versus SFB-positive (y axis) NOD mice 6–10 wk of
age. Highlighted are several genes characteristic of Th17 cells (red type),
other CD4+T-cell subsets (blue type), or genes located within the idd3 di-
abetes susceptibility interval (orange type). Values in the upper left and
lower right corners refer to the numbers of loci up- or down-regulated,
respectively, by more than twofold. (B) As in A, except that values for co-
lonic lamina propria transcripts are plotted. (C) An expression heatmap for
transcription factors and cytokines diagnostic of Th17 cells and other CD4+
T-cell subsets. Data are from SI-LP or splenic CD4+T cells from NOD mice that
were positive or negative for SFB. All mice were 6–10 wk of age. Data are
Specific induction of Th17 transcripts in SI-LP CD4+T cells of SFB-
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| www.pnas.org/cgi/doi/10.1073/pnas.1108924108Kriegel et al.
When considering a possible mechanism for SFB-mediated
protection from NOD diabetes, a role for Th17 cells immediately
comes to mind. De novo introduction of SFB into mice in sever-
al experimental contexts quite specifically promoted the de-
velopment of a robust Th17 compartment in the SI-LP (6, 17)
[although one group did observe a broader influence on Th
subsets (5)], and the impact of these cells was translated distally
to promote nongut autoimmune diseases (17, 18). The function of
IL-17–producing cells in NOD diabetes has been a topic of much
debate. Some investigators have argued for a critical role, in
particular in later, effector processes (36–38), although others
have cautioned about possible misinterpretations of findings from
Th17 transfer experiments because of the conversion to or ex-
pansion of Th1 cells (39, 40). In clear contradiction, IL-17–
producing cells were found either to inhibit diabetes in NOD
mice, as well as in BioBreeding rats, in several experimental
contexts (41–43) or to have no effect (44). These divergent con-
clusions likely reflect multiple complexities: that different Th
subsets may play variable roles according to the particular disease
stage; that multiple cell types can produce IL-17; and that Th17
cells produce cytokines other than IL-17. Nonetheless, an at-
tractive hypothesis to pursue is that SFB colonization of NOD
mice induces the emergence of a robust Th17 population in the
gut, and that this population can impact diabetogenesis by
inhibiting the islet-directed Th1 response, a notion consistent
with a recent report that Th17 cells inhibit Th1 effector cells in
a colitis model (45). Because insulitis appeared to be typical in 10-
wk-old SFB-positive females, Th17 cells would not influence
disease triggering but rather would dictate the “flavor” of the
insulitis that sets in and/or its evolution to overt diabetes.
It also is possible that Th17-produced mediators other than
the signature cytokine IL-17A are responsible for SFB-promoted
protection from NOD diabetes. For example, IL-22, a member
of the IL-10 cytokine family, is thought to confer transepithelial
resistance to injury (as reviewed in refs. 46, 47). Breach of the
gastrointestinal barrier with subsequent leakage of potentially
immunogenic dietary components has been proposed to promote
T1D in NOD mice (48) and is a well-established trigger of celiac
disease, an anti–gliadin-directed autoimmune disorder that fre-
quently accompanies T1D in humans (49, 50). Moreover, we
previously demonstrated that gut irritation in NOD mice alters
the priming of diabetogenic islet-derived self-antigens in the
PLNs (31). It is possible that, in the absence of SFB, other gut
microbes provoke intestinal stress in female NOD mice; SFB-
induced Th17 cells might produce mediators, such as IL-22, that
are beneficial for epithelial barrier integrity. The unusually in-
timate interactions of SFB with the gut epithelium might be
another element impacting the gut barrier (51).
Interestingly, SFB was not primarily responsible for the low
incidence of diabetes in male NOD mice in our colony: Both
SFB-positive and SFB-negative males had a low incidence of
<20%. Because a substantially higher penetrance is achievable in
other colonies (in particular GF colonies) (e.g., ref. 24), we
suspect that another microbe (or other microbes) might be re-
sponsible. A different downstream mechanism probably is in-
volved, because SFB-negative males and females had similarly
low levels Th17 cells in the SI-LP (and elsewhere), and Th17
cells and signature transcripts were similarly induced in SFB-
positive mice of both sexes. A comparison of the male and fe-
male gastrointestinal microbiomes via high-throughput sequenc-
ing should prove informative in this regard.
In conclusion, we have demonstrated that cosegregation of
diabetes protection with a single gut-resident commensal can
explain variable penetrance of an organ-specific autoimmune
disease in different mouse colonies. It is likely that patterns of
colonization by immunomodulatory commensals also are at play
in other immunologic disease models with varying penetrance.
Finally, we speculate that the development of autoimmune dis-
ease in genetically predisposed humans also might be strongly
influenced by commensal colonization patterns, especially con-
sidering that microbiota represent a rich set of “environmental
modifiers,” with more than 100 trillion bacteria persistently col-
onizing our mucosal surfaces (52). SFB itself has not yet been
identified in humans, but the possibility that another microbe fills
its gut niche remains unexplored.
Materials and Methods
Mice. B6, BALB/c, and NOD mice were purchased from TAC or JAX or were
housed in our animal facility at the NRB. Details of strains and their handling
are given in SI Materials and Methods.
Tissue Collection and DNA Isolation. Procedures entailed in feces collection,
dissection of intestinal segments, lymphoid tissue preparation, and genomic
DNA isolation are detailed in SI Materials and Methods.
SFB Quantification. Genomic DNA from fecal pellets or tissues was amplified
with SFB-specific [SFB736F and SFB884R (53)] and conserved EUB 1114F and
1221R (54) 16S ribosomal DNA primers. Primer sequences are listed in SI
Materials and Methods. The ratio of the EUB/SFB values, calculated as in ref. 6,
yielded relative SFB levels.
Diabetes and Insulitis Assessments. Mice were screened weekly for diabetes
development as described in SI Materials and Methods. Insulitis was assessed
at 10 wk of age as detailed in SI Materials and Methods.
Preparation of Intestinal Cell Suspensions for Immunologic Analyses. Cell sus-
pensions were prepared from the SI-LP and colon lamina propria of 6- to 10-
wk-old NOD mice as recently described (17) and as detailed in SI Materials
and Methods. Cell preparations from the spleen and PLN were by standard
procedures, as described in ref. 31.
Flow Cytometry. Flow cytometric analyses of lymphocytes from the small
intestine, colon, spleen, and PLN were performed as described previously (17,
55) and are explained in more detail in SI Materials and Methods.
Microarray Analysis. Cells were sorted, RNA was isolated and hybridized to
GeneChip Mouse Gene 1.0 ST arrays (Affymetrix), and data were analyzed as
described in SI Materials and Methods. Datasets are available at the National
Center for Biotechnology Information (accession no. 29806).
Statistical Analyses. Data are presented as mean ± SEM. Statistical signifi-
cance was assessed using the student’s t test, Mann–Whitney test, or Pearson
correlation, as indicated in the figure legends. The log-rank test was used for
the cumulative incidence analyses. P values <0.05 were considered to be
ACKNOWLEDGMENTS. We thank A. Wilcox, K. Hattori, and A. Ortiz-Lopez
for assistance with mice; K. Leatherbee and J. Ericson for help with
microarrays; and C. Laplace for figure preparation. This work was supported
by Grant 5R01 DK59658 from the National Institutes of Health and by
a generous gift from the Howalt family to D.M. and C.B. J.A.H. was
supported by a postdoctoral fellowship from the Canadian Institutes of
Health Research and the Canadian Diabetes Association. H.-J.W. was
supported by a grant from the Arthritis National Research Foundation.
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