Microbiota innate stimulation is a prerequisite for T cell spontaneous proliferation and induction of experimental colitis.

Abstract

Little is known about how the microbiota regulates T cell proliferation and whether spontaneous T cell proliferation is involved in the pathogenesis of inflammatory bowel disease. In this study, we show that stimulation of innate pathways by microbiota-derived ligands and antigen-specific T cell stimulation are both required for intestinal inflammation. Microbiota-derived ligands promoted spontaneous T cell proliferation by activating dendritic cells (DCs) to produce IL-6 via Myd88, as shown by the spontaneous proliferation of T cells adoptively transferred into specific pathogen-free (SPF) RAG-/- mice, but not in germfree RAG-/- mice. Reconstitution of germfree RAG-/- mice with cecal bacterial lysate-pulsed DCs, but not with IL-6-/- or Myd88-/- DCs, restored spontaneous T cell proliferation. CBir1 TCR transgenic (CBir1 Tg) T cells, which are specific for an immunodominant microbiota antigen, induced colitis in SPF RAG-/- mice. Blocking the spontaneous proliferation of CBir1 Tg T cells by co-transferring bulk OT II CD4+ T cells abrogated colitis development. Although transferred OT II T cells underwent spontaneous proliferation in RAG-/- mice, the recipients failed to develop colitis because of the lack of cognate antigen in the intestinal lumen. Collectively, our data demonstrate that induction of colitis requires both spontaneous proliferation of T cells driven by microbiota-derived innate signals and antigen-specific T cell proliferation.

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Article
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 207 No. 6 1321-1332
www.jem.org/cgi/doi/10.1084/jem.20092253
1321
The pathogenesis of inflammatory bowel dis-
ease (IBD) involves the presence of one or
more genetically determined defects that result
in an abnormal mucosal immune system that
overreacts to normal constituents of the micro-
biota (Elson et al., 2005; Sartor, 2006; Strober
et al., 2007). The complex intestinal microbiota
provides an abundant source of immunostimula-
tory organisms containing innate stimulatory
materials and antigens that can activate innate
and adaptive immune responses, respectively.
In multiple experimental models of colitis, the
microbiota plays a crucial role, in that the mice
develop colitis with a conventional microbiota
but fail to develop colitis when housed under
germfree conditions (Taurog et al., 1994; Rath
et al., 1996; Sellon et al., 1998; Frank et al.,
2007). It is now generally accepted that com-
mensal enteric bacteria provide constant anti-
genic stimulation that can activate pathogenic
T cells and lead to chronic intestinal inflamma-
tion. Data from our own studies and others
suggest that an excessive adaptive immune re-
sponse to gut microflora antigens is indispens-
able for intestinal inflammation (Lodes et al.,
2004; Xavier and Podolsky, 2007). Transfer of
CD4+ T cells reactive to a single commensal
flagellin antigen or to a single bacteria-associated
antigen is able to induce colitis development
in immunodeficient mice (Iqbal et al., 2002;
Lodes et al., 2004; Watanabe et al., 2006). All
the subsets of CD4+ effector T cells reactive to
commensal bacterial antigens have been impli-
cated in the pathogenesis of IBD. IFN-–Th1
and IL-23/IL-17–Th17 pathways are associated
CORRESPONDENCE
Yingzi Cong:
yicong@utmb.edu
Abbreviations used: ASF,
Altered Schaedler’s Flora;
BMDC, BM-derived dendritic
cell; CBL, cecal bacterial lysate;
IBD, inflammatory bowel disease;
LP, lamina propria; SPF, specific
pathogen-free; STAT, signal
transducer and activator of
transcription; TET, Tetanus
toxin fragment C; Tg, transgenic;
TLR, Toll-like receptor.
Microbiota innate stimulation
is a prerequisite for T cell spontaneous
proliferation and induction
of experimental colitis
Ting Feng,1 Lanfang Wang,2 Trenton R. Schoeb,3 Charles O. Elson,1,2
and Yingzi Cong1,2
1Department of Microbiology, 2Division of Gastroenterology and Hepatology, Department of Medicine, and 3Department
of Genetics, University of Alabama at Birmingham, Birmingham, AL 35294
Little is known about how the microbiota regulates T cell proliferation and whether
spontaneous T cell proliferation is involved in the pathogenesis of inflammatory bowel
disease. In this study, we show that stimulation of innate pathways by microbiota-derived
ligands and antigen-specific T cell stimulation are both required for intestinal inflammation.
Microbiota-derived ligands promoted spontaneous T cell proliferation by activating
dendritic cells (DCs) to produce IL-6 via Myd88, as shown by the spontaneous proliferation
of T cells adoptively transferred into specific pathogen–free (SPF) RAG/ mice, but not in
germfree RAG/ mice. Reconstitution of germfree RAG/ mice with cecal bacterial
lysate–pulsed DCs, but not with IL-6/ or Myd88/ DCs, restored spontaneous T cell
proliferation. CBir1 TCR transgenic (CBir1 Tg) T cells, which are specific for an immuno-
dominant microbiota antigen, induced colitis in SPF RAG/ mice. Blocking the spontaneous
proliferation of CBir1 Tg T cells by co-transferring bulk OT II CD4+ T cells abrogated colitis
development. Although transferred OT II T cells underwent spontaneous proliferation in
RAG/ mice, the recipients failed to develop colitis because of the lack of cognate antigen
in the intestinal lumen. Collectively, our data demonstrate that induction of colitis requires
both spontaneous proliferation of T cells driven by microbiota-derived innate signals and
antigen-specific T cell proliferation.
© 2010 Feng et al. This article is distributed under the terms of an Attribution–
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the publication date (see http://www.rupress.org/terms). After six months it is
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by-nc-sa/3.0/).
The Journal of Experimental Medicine

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Microbiota innate stimulation in colitis | Feng et al.
In this study, we investigated the effect of the microbiota
on T cell spontaneous proliferation and the role of T cell
spontaneous proliferation in the pathogenesis of intestinal in-
flammation. A mouse TCR transgenic line (CBir1 Tg) which
is specific for the immunodominant microbiota flagellin anti-
gen, CBir1, present in the intestinal lumen (Cong et al.,
2009), as well as a TCR transgenic line (OT II) specific for
ovalbumin which is not present in the intestinal lumen was
used. We demonstrate that the microbiota provided innate
stimulatory activity to promote T cell spontaneous prolifera-
tion by activating DCs to produce IL-6 via TLR–TLR li-
gand interaction through a Myd88-dependent pathway. Both
microbiota innate stimulation-driven T cell spontaneous pro-
liferation and microbiota-induced antigen-specific T cell ac-
tivation were required for the induction of colitis.
RESULTS
Transfer of CBir1 Tg CD4+ T cells, but not OT II CD4+ T cells,
induces colitis in B6.RAG/ mice under SPF conditions
To determine the role of commensal bacterial antigens in in-
duction of colitis in lymphopenic mice, 106 CD4+ T cells
from B6.CBir1 Tg mice specific for a microbiota flagellin
present in intestinal lumen and CD4+ T cells from B6.OT II
mice specific for OVA were separately transferred into spe-
cific pathogen–free (SPF) B6.RAG/ mice. RAG/ re-
cipients of CBir1 Tg CD4+ T cells, but not OT II CD4+
T cells, lost weight starting from week 3 (Fig. 1 A). All recipi-
ents that received CBir1 Tg CD4+ T cells developed severe
colonic inflammation when sacrificed 8 wk after cell transfer.
In contrast, none of the recipients that received OT II CD4+
T cells developed colitis (Fig. 1, B–D), indicating that activa-
tion by cognate antigen in the intestine plays a crucial role in
the induction of colitis. Both CBir1 Tg CD4+ T cells and
OT II CD4+ T cells proliferated, and substantial numbers of
CD4+ T cells were recovered from the intestinal lamina pro-
pria (LP) of recipients. Notably, more LP CD4+ T cells were
recovered from the RAG/ recipients of CBir1 Tg CD4+
T cells (3.2 × 105/mouse) than from the recipients of OT II
CD4+ T cells (1 × 105/mouse). Th1 and Th17 cells have been
implicated in the induction of colitis in various animal
models of colitis and in patients with IBD (Cong et al., 2002;
Iqbal et al., 2002; Hue et al., 2006; Kullberg et al., 2006;
Elson et al., 2007; Kobayashi et al., 2008; Brand, 2009;
Zenewicz et al., 2009). To determine if there is different cy-
tokine production in the recipients reconstituted with CBir1
Tg CD4+ T cells vs. with OT II CD4+ T cells, LP CD4+
T cell production of IFN- and IL-17 was determined by
flow cytometry 8 wk after cell transfer. LP CD4+ T cells from
the recipients of OT II T cells produced IFN- (8.69%) and
IL-17 (4.14%) at a low frequency, whereas LP CD4+ T cell
from the recipients of CBir1 Tg T cells produced IFN-
(21.2%) and IL-17 (17.0%) at a much higher frequency. In-
terestingly, a substantial proportion of IL-17+IFN-+ double
positive LP CD4+ T cells were present in colitic RAG/
recipient mice reconstituted with CBir1 Tg T cells but not
in OT II T cell reconstituted recipients (Fig. 1 E). Whether
with Crohn’s disease, whereas the IL-13–Th2 pathway is as-
sociated with ulcerative colitis. It has also been shown that
microbial components are necessary for the onset of abnor-
mal inflammatory responses because of their capacity to
stimulate innate cells via membrane-bound and endosomal
Toll-like receptors (TLRs). TLR4 deficiency abrogates colitis
development in myeloid-specific signal transducer and acti-
vator of transcription (STAT)-3/ mice (Kobayashi et al.,
2003) and Myd88 deficiency blocks colitis development in
IL-10–deficient mice (Rakoff-Nahoum et al., 2006). How-
ever, the mechanism by which the Myd88 pathway supports
intestinal inflammation is still largely unknown. It is also un-
known whether microbiota stimulation of innate responses
via TLR ligands and adaptive immune responses to microbi-
ota antigens contributes to intestinal inflammation indepen-
dently or whether they interact with another.
Homeostatic mechanisms control the overall size and
composition of the mature T cell pool to keep the overall
number of T cells relatively constant during life (Jameson,
2002; Min et al., 2005; Surh and Sprent, 2008). Accumu-
lating data indicate a role for T cell homeostatic proliferation
in the pathogenesis of autoimmune diseases (Theofilopoulos
et al., 2001; King et al., 2004; Krupica et al., 2006; Milner
et al., 2007; Chang et al., 2008; McPherson et al., 2009), as
well as in controlling HIV reservoir size and persistence in
latently infected CD4+ T cells (Chomont et al., 2009). In
response to lymphopenia, naive T cells proliferate and tran-
sition into a memory-like state through a process termed
“homeostatic proliferation” (Jameson, 2002). T cell homeo-
stasis is regulated by two distinct modes of cell prolifera-
tion: slow IL-7/IL-15-dependent homeostatic proliferation
and rapid MHC/antigen interaction-dependent spontane-
ous proliferation (Schluns et al., 2000; Ku et al., 2000;
Goldrath et al., 2002, 2004; Min et al., 2004). A recent
work demonstrates that IL-6 is responsible for spontane-
ous proliferation in an adoptive transfer model of colitis,
in that blockade of IL-6 inhibited CD8+ T cell spontane-
ous proliferation as well as colitis development in RAG/
recipient mice (Tajima et al., 2008). However, the factors
that stimulate IL-6 production, the cellular source of IL-6,
and the exact role of IL-6 in T cell spontaneous prolifera-
tion are still unknown. The intestinal microbiota has been
shown to be required for T cell spontaneous proliferation.
Germfree mice have underdeveloped mucosal and systemic
immune systems with decreased cellularity of gut associ-
ated lymphoid tissues, mesenteric lymph nodes, and spleen
(Cebra, 1999; Mazmanian et al., 2008). Adoptively trans-
ferred polyclonal CD4+ T cells do not undergo spontaneous
proliferation in germfree SCID mice but do in conven-
tionally reared SCID mice (Kieper et al., 2005). However,
it is unclear whether the microbiota-driven T cell spontane-
ous proliferation is caused by microbiota antigenic stimulation
or their interaction with host innate cells via TLR–TLR
ligand interactions, and whether microbiota-driven T cell
spontaneous proliferation is required for the induction
of colitis.

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JEM VOL. 207, June 7, 2010
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such IL-17+IFN-+ CD4+ T cells are the pathogenic T cells
that drive colitis development is unclear at present.
To investigate whether lack of colitis induction in the
RAG/ recipient mice that received OT II T cells was
caused by significantly less proliferation of OT II T cells than
CBir1 Tg T cells upon transfer into RAG/ mice and inde-
pendent of antigen recognition, 5 × 106 naive or OT II
CD4+ T cells that had been activated with OVA for a week
were transferred into SPF B6.RAG/ mice, and the recipi-
ent mice were then colonized with OVA-expressing Esche-
richia coli (OVA-E. coli) or irrelevant Tetanus toxin fragment
C (TET)–expressing E. coli (TET-E. coli). When sacrificed
8 wk later, neither naive nor preactivated OT II T cells
induced colitis in the recipients colonized with TET-
expressing E. coli (Fig. 1 F), but all recipients colonized with
OVA-expressing E. coli did develop colitis with a pathology
score of 64 ± 21 (Fig. 1 G), indicating that lack of colitis in
the OT II T cell recipients was not caused by the number of
OT II T cells, but by lack of cognate antigen in the colon.
Collectively, these data demonstrated a crucial role of bacte-
rial-associated antigens in the induction of colitis.
To determine whether the inability of OT II T cells to
induce colitis in the RAG/ recipients is caused in part by
the lack of homeostatic proliferation and/or spontaneous
proliferation of OT II T cells in RAG/ mice, CBir1 Tg
CD4+ T cells and OT II CD4+ T cells were labeled with
CFSE and transferred separately into RAG/ mice. As
shown in Fig. 2, both CBir1 Tg CD4+ T cells and OT II
CD4+ T cells underwent homeostatic proliferation and spon-
taneous proliferation. Approximately 43% of OT II T cells
Figure 1. Adoptive transfer of CBir1 Tg
CD4+ T cells but not OT II CD4+ T cells
induces colitis in RAG/ mice. Groups of 5
B6.RAG/ mice were injected with 106 CBir1
Tg CD4+ T cells, 106 OT II CD4+ T cells, or PBS
as control. Recipient mice were weighed
weekly. 8 wk after transfer, the mice were
sacrificed and assessed for histopathology.
LP CD4+ T cell cytokine production was
determined by flow cytometry. (A) Weight
changes of the recipient mice. *, P < 0.05
compared with the PBS group. (B) Pathologi-
cal scores of the recipient mice. **, P < 0.01
compared with the PBS group. (C and D) Co-
lonic histopathology of the recipient mice.
(E) Intestinal LP CD4+ T cell IL-17 and IFN-
production. (F and G) Groups of 5 B6.RAG/
mice were injected with 5 × 106 OT II CD4+ T
cells and then intrarectally administered with
TET-E. coli (F) or with OVA-E. coli (G) every
week. Data are from one of two independent
experiments with similar results. Bars: (C, D,
and F) 50 μm; (E) 100 μm.
Figure 2. More CBir1 Tg CD4+ T cells undergo spontaneous prolif-
eration than do OT II CD4+ T cells in RAG/ mice. 106 CFSE-CBir1 Tg
CD4+ T cells or 106 CFSE-OT II CD4+ T cells were transferred into RAG/
mice, and the recipient mice were sacrificed 10 d later. CD4+ T cell prolifera-
tion in the spleen was analyzed by CFSE dilution by flow cytometry. (A) OT II
CD4+ T cell proliferation in RAG/ mice. (B) CBir1 Tg CD4+ T cell prolifera-
tion in RAG/ mice. Numbers in each plot represent the percentage of
donor cells undergoing spontaneous proliferation, homeostatic prolifera-
tion, and staying undivided, respectively. Data are representative of at least
four individual mice of each group from three independent experiments.
(C) Percentage of transferred T cells in spontaneous proliferation (SP), ho-
meostatic proliferation (HP), and undivided from three independent experi-
ments. *, P < 0.05 for CBir1 Tg T cells against OT II T cells.

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Microbiota innate stimulation in colitis | Feng et al.
proliferation was determined by flow
cytometry based on CFSE dilution.
As previously shown, CBir1 Tg CD4+
T cells and OT II CD4+ T cells un-
derwent both homeostatic prolifera-
tion and spontaneous proliferation in
SPF RAG/ mice (Fig. 3, A and F),
whereas both TCR Tg T cells under-
went only homeostatic proliferation, but not spontaneous
proliferation, in RAG/ mice housed in germfree condi-
tions (Fig. 3, B and G). Interestingly, both TCR Tg T
cells underwent homeostatic proliferation and spontaneous
proliferation in RAG/ mice reconstituted with Altered
Schaedler’s Flora (ASF; Fig. 3, C and H). As previously
shown, CBir1 Tg T cells induced colitis in SPF RAG/
mice, but not in germfree RAG/ mice, 8 wk after cell
transfer (Fig. 3, D and E). These data are consistent with an-
tigenic stimulation by the microbiota driving T cell sponta-
neous proliferation, as previously suggested (Kieper et al.,
2005; Surh and Sprent, 2008); however, they do not exclude
a role for microbiota innate stimulation in T cell spontane-
ous proliferation.
Microbiota stimulation of DC IL-6 production via Myd88
promotes T cell spontaneous proliferation
To determine whether microbiota-DC interactions affect
T cell homeostatic proliferation and/or spontaneous prolifer-
ation, RAG/.OT II CD4+ T cells were transferred into
germfree RAG/ mice together with BM-derived DCs
(BMDCs) that were or were not pulsed with cecal bacterial
lysates (CBLs) from conventional SPF mice. RAG/.OT II
CD4+ T cells underwent only homeostatic proliferation in
the RAG/ recipients reconstituted with control PBS-
pulsed BMDCs (Fig. 4 A). However, they underwent both
underwent spontaneous proliferation, 11.8% underwent homeo-
static proliferation, and 45.3% remained undivided (Fig. 2,
A and C). A higher proportion of CBir1 Tg CD4+ T cells
underwent spontaneous proliferation (Fig. 2, B and C). Approx-
imately 72.9% of CBir1 Tg T cells underwent spontaneous
proliferation, 6.18% underwent homeostatic proliferation, and
20.9% stayed undivided (Fig. 2 B). This could be caused by
differences of TCR affinity between OT II T cells and CBir1
Tg T cells, or by the presence of CBir1 flagellin, but not OVA,
in the intestine, which could further stimulate CBir1 Tg T cell
spontaneous proliferation.
CD4+ T cells do not undergo spontaneous proliferation
in germfree RAG/ mice
In multiple experimental colitis models, mice do not develop
colitis under germfree conditions. We next asked whether
microbiota could affect colitis induction via regulation of
T cell spontaneous proliferation. As reported previously
(Cebra, 1999; Mazmanian et al., 2008), the total number of
splenic CD4+ and CD8+ T cells in germfree mice are only
80% of that of conventional SPF mice, indicating a mild
lymphopenic condition. To further investigate the role of
intestinal microbiota in T cell spontaneous proliferation,
CFSE-labeled CBir1 Tg CD4+ T cells or OT II CD4+ T cells
were transferred into RAG/ mice housed in conventional
SPF conditions or in germfree conditions. 10 d later, T cell
Figure 3. CD4+ T cells do not undergo
spontaneous proliferation in germfree
RAG/ mice. 106 CFSE-CBir1 Tg CD4+ T cells
(A–C) or 106 CFSE-OT II CD4+ T cells (F–H)
were transferred into RAG/ mice housed
under SPF conditions (A and F), germfree (GF)
conditions (B and G), or ASF conditions (C and
H). 10 d after the transfer, division of the
CFSE-labeled CD4+ T cells was determined by
CFSE dilution. Numbers in each plot represent
the percentage of donor cells undergoing
spontaneous proliferation, homeostatic prolif-
eration, and staying undivided, respectively.
Data are representative of at least three indi-
vidual mice of each group from three indepen-
dent experiments. Histopathology of the colon
of CBir1 Tg T cell recipients at 5 mice/group
under SPF conditions (D) and germfree condi-
tions (E) was assessed 8 wk after cell transfer.
Bars, 100 µm. Data are representative of three
independent experiments with similar results.

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JEM VOL. 207, June 7, 2010
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they only underwent homeostatic prolifera-
tion but not spontaneous proliferation in the
recipients reconstituted with CBL-pulsed
IL-6/ BMDCs (Fig. 4 D), demonstrating
that DC IL-6 production plays a crucial role
in the CD4+ T cell spontaneous proliferation
driven by the microbiota.
Multiple microbial products stimulate DC cytokine pro-
duction via TLR–TLR ligand interaction through Myd88
(Medzhitov et al., 1998; Beutler, 2004; Akira et al., 2006;
Medzhitov, 2007). To determine the role of Myd88 pathway
in microflora-driven CD4+ T cell spontaneous proliferation
in vivo, RAG/.OT-II CD4+ T cells were transferred
into germfree RAG/ mice together with CBL-pulsed
Myd88/ BMDCs or control CBL-pulsed WT BMDCs.
As shown previously, CD4+ T cells underwent homeostatic
and spontaneous proliferation in the germfree RAG/ re-
cipient mice reconstituted with CBL-pulsed WT BMDCs.
However, in the RAG/ recipient mice reconstituted with
CBL-pulsed Myd88/ BMDCs, RAG/.OT II T cells
only underwent homeostatic proliferation, but not spontaneous
proliferation (Fig. 4 E), confirming that the Myd88-
dependent TLR pathway plays a crucial role in microbiota-
driven CD4+ T cell spontaneous proliferation through the
induction of DC IL-6 production.
homeostatic proliferation and spontaneous proliferation in the
RAG/ recipients reconstituted with CBL-pulsed BMDCs
(Fig. 4 B), indicating that microbiota-stimulated DCs can
result in nonspecific stimulation of CD4+ T cell spontaneous
proliferation.
IL-6 has been shown to be a vital cytokine in CD8+
T cell spontaneous proliferation in a recent study (Tajima
et al., 2008). We next determined the role of IL-6 in the
promotion of CD4+ T cell spontaneous proliferation by CBL-
pulsed DCs. CBL-pulsed BMDCs produced high amounts
of IL-6, as well as TNF and IL-1 in vitro, and such IL-6 and
other cytokine production was impaired in CBL-pulsed
Myd88/ BMDCs, indicating that the Myd88 is involved
in CBL-stimulated DC IL-6 production (Fig. 4 C). RAG/.
OT II CD4+ T cells were then transferred into germfree
RAG/ mice together with WT or IL-6/ CBL-pulsed
BMDCs. RAG/.OT II CD4+ T cells underwent both ho-
meostatic proliferation and spontaneous proliferation in the
recipients reconstituted with CBL-pulsed WT BMDCs, whereas
Figure 4. Microbiota stimulation of DC IL-6
production via Myd88 pathway promotes T cell
spontaneous proliferation. BMDCs generated from
B6 mice were pulsed with PBS or 100 µg/ml of CBLs
for 24 h. 106 CFSE-RAG/.OT II CD4+ T cells were
transferred together with 106 PBS-pulsed BMDCs (A)
or with CBL-pulsed BMDCs (B) into germfree RAG/
mice. 10 d after the transfer, division of the CFSE-
labeled CD4+ T cells was determined by flow cytom-
etry. Numbers in each plot represent the percentage
of donor cells undergoing spontaneous proliferation,
homeostatic proliferation, and staying undivided,
respectively. (C) BMDCs generated from B6 mice or
B6.Myd88/ mice were stimulated with 100 µg/ml
of CBL for 24 h. To determine IL-1 production, 2
mM ATP was added in the last 10 min of the culture.
IL-6, TNF, and IL-1 production in the supernatant
was determined by ELISA. **, P < 0.01 compared with
WT DCs. (D) 106 CFSE-RAG/.OT II CD4+ T cells were
transferred together with 106 CBL-pulsed B6.BMDCs
or CBL-pulsed B6.IL-6/ BMDCs into germfree
RAG/ mice. (E) 106 CFSE-RAG/.OT II CD4+ T cells
were transferred together with 106 CBL-pulsed
B6.BMDCs or CBL-pulsed B6.Myd88/ BMDCs into
germfree RAG/ mice. 10 d after the transfer,
division of the CFSE-labeled CD4+ T cells was
determined by flow cytometry. Numbers in each plot
represent the percentage of donor cells undergoing
spontaneous proliferation, homeostatic proliferation,
and undivided, respectively. Data are representative
of at least four mice of each group from two
independent experiments.