of June 18, 2013.
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Tropism of Inflammatory Th17 Cells in the
Retinoic Acid Determines the Precise Tissue
Love and Chang H. Kim
Chuanwu Wang, Seung G. Kang, Harm HogenEsch, Paul E.
2010; 184:5519-5526; Prepublished online 16
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The Journal of Immunology
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The Journal of Immunology
Retinoic Acid Determines the Precise Tissue Tropism of
Inflammatory Th17 Cells in the Intestine
Chuanwu Wang,* Seung G. Kang,* Harm HogenEsch,* Paul E. Love,†,‡and
Chang H. Kim*
Th17cells are majoreffectorT cells in theintestine,but theregulation of their tissuetropism within thegutis poorly understood.We
investigated the roles of vitamin A and retinoic acid in generation ofinflammatory Th17 cells with distinct tissue tropisms within the
intestine. We found that Th17 cells with distinct tissue tropisms and pathogenic activities are generated depending on the available
concentration of retinoic acid (RA). In contrast to the widespread perception that RAwould suppress the generation of Th17 cells,
we provide evidence that RA is actually required for generation of Th17 cells with specific tissue tropisms within the gut. Th17 cells
the Th17 cells induced with optimal levels of exogenous RA (∼10 nM) migrated to the small intestine and induced more severe
inflammation. The Th17 cells, induced in the presence or absence of RA, differentially expressed the trafficking receptors CCR9
and a4b7. CCR9 is required for Th17 cell migration to the small intestine, whereas a4b7 is required for the migration of Th17 cells
combination of trafficking receptors regulated by the RA signal.
groups of intestinal commensal bacteria provide the major signal
for such enrichment of Th17 cells (3, 4). Although it is thought that
Th17 cells are major effector T cells that provide immunity against
potential pathogens (5, 6), the exact roles of Th17 cells in the in-
testine are incompletely understood. Th17 cells can induce or
regulate the inflammation in the gut. Th17 cell cytokines are in-
creased invarious types of inflammatory bowel diseases (7–9), and
these cytokines have been implicated in the pathogenesis of in-
flammatory bowel diseases (1, 2, 10, 11). In contrast, another Th17
cell cytokine IL22 has a protective role in colitis (12). It is not
well understood how Th17 cells acquire specific tissue tropisms
and segment-specific inflammatory activity within the intestine.
The intestine absorbs and metabolizes vitamin A into retinoic
environment (13–17). In this regard, RA is particularly associated
with imprinting the tissue tropism of T cells and B cells for their
migration into the small intestine (18–20). It has been reported
that RA can suppress the differentiation of naive T cells into Th17
The Journal of Immunology, 2010, 184: 5519–5526.
helper 17 are highly enriched in the intestinal lamina
propria (1). They are present in all segments of the in-
testine, including the largeand small intestine (2). Certain
cells, at least in vitro (19, 21–24). Together with its function in
inducing FoxP3+regulatory T cells, RA is considered a key reg-
ulatory signal in inducing immune tolerance in the intestine (25).
We investigated the role of RA in generation of Th17 cells with
distinct tissue tropisms within the intestine. RA at physiologically
relevant concentrations induced gut homing Th17 cells which are
highly inflammatory upon transfer into mice. These cells were
particularly active in migrating and inducing inflammation in the
suboptimal concentrations of RA, induced inflammation mainly in
the large intestine. We also report that the two gut-trafficking
receptors CCR9 and a4b7 play distinctive roles in migration and
function of the retinoid-induced Th17 (RATh17) cells in eliciting
inflammation in the intestine. Therefore, our results establish that
distinct Th17 cell subsets with specific tissue tropism are generated
depending on the available concentrations of RA. Our findings are
important in understanding the origin, trafficking, and function of
Th17 cells in the intestine.
Materials and Methods
Cell isolation and culture
Naive CD4+T cells were isolated from pooled mononuclear cells of pe-
ripheral lymph nodes, mesenteric lymph nodes (MLNs), and spleens as
described before (2). Naive CD4+T cells were cultured in complete RPMI
1640 medium (10% FBS) supplemented with Con A (2.5 mg/ml), hTGF-b1
(10 ng/ml; PeproTech), mIL-23 (10 ng/ml; R&D Systems, Minneapolis,
mIL-4 (11B11, 10 mg/ml; BioLegend, San Diego, CA), anti–mIFN-g
(XMG2.4, 10 mg/ml; BioLegend) and anti–mIL-2 (S4B6, 2.5 mg/ml; BD
Biosciences, San Jose, CA) for 7 d to generate control Th17 cells that were
cells were prepared using the same condition with RA at physiologic con-
centrations (10–20 nM). In some experiments, naive T cells were CFSE-
labeled and cultured in the Th17 cell induction condition to determine the
relationship between cell division and expression of the gut-trafficking re-
ceptors (Supplemental Fig. 1). The Th17 cells were also recultured for an
additional 7 d to determine the stability of trafficking receptor expression
(Supplemental Fig. 2).
*Laboratory of Immunology and Hematopoiesis, Department of Comparative Patho-
biology, Purdue Cancer Center, Purdue University, West Lafayette, IN 47907; and
and Human Development and‡Laboratory of Clinical Investigation, National Institute
of Allergyand Infectious Diseases,NationalInstitutes ofHealth, Bethesda, MD 20892
Received for publication December 9, 2009. Accepted for publication March 14,
This work was supported by National Institutes of Health Grants 1R01AI074745,
1R56AI080769, and 1R01DK076616 and the Crohn’s and Colitis Foundation of
America (to C.H.K.).
Address correspondence and reprint requests to Dr. Chang Kim, Department of
Comparative Pathobiology, 725 Harrison Street, Purdue University, West Lafayette,
IN 47907. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this paper: Itg, integrin; MLN, mesenteric lymph node; PP,
Peyer’s patch; RA, retinoic acid; RA Th17, retinoid-induced Th17; TRITC, tetra-
by guest on June 18, 2013
Animals and generation of vitamin A-deficient or -sufficient mice
All the experiments with animals in this study were approved by the Purdue
University Animal Care and Use Committee. CCR9-deficient mice were
described previously (26). Integrin (Itg) b7-deficient mice (C57BL/6-
Itgb7tm1Cgn/J) and Rag1-deficient (B6.129s7-Rag1tm1Mom/J) mice were
purchased from The Jackson Laboratory (Bar Harbor, ME). AKR/J mice
93G containing 25,000 IU/kg or 0 IU/kg levels of retinyl acetate (Harlan
Teklad TD-06528 and 07267) immediately following birth. The pups were
weanedat 4 wk oldand maintained on the same dietsfor anadditional9 wk.
Vitamin A deficiency was verified by determining defective CCR9 expres-
sion by small intestinal T cells, as described previously (2).
a Th17 cell induction condition for 7 d at the indicated concentrations of RA. B, Expression of gut homing receptors (CCR9 and a4b7) by Th17 cells was
examined. C, The chemotactic ability of RATh17 cells and control Th17 cells to CCL25 and CCL19. D, Twenty-hour, short-term homing capacity of RA
Th17 cells and control Th17 cells was compared in C57BL/6 mice. The Th17 cells were injected i.v. Homing index of RATh17 cells is shown as percent of
control Th17 cells. *A significant increase in migration of RA Th17 cells over control Th17 cells. E, Migration of RA Th17 cells is shown as percent
distribution among the indicated organs.pSignificant differences from control Th17 cells. F, In situ visualization of the RA Th17 cells and control Th17
cells migrated into indicated organs (original magnification 3200). All experiments were performed at least three times and combined (A–E) or repre-
sentative data (F) are shown. Averages and SEM are shown in the combined data.
Gut homing CCR9+a4b7+Th17 cells are induced in the presence of physiological concentrations of RA. A, Naive T cells were cultured in
5520RETINOIC ACID AND TISSUE TROPISM OF INTESTINAL Th17 CELLS
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Flow cytometry to identify Th17 cells and to determine their
expression of trafficking receptors
Intracellular cytokine staining for IL-17 (TC11-18H10.1; BioLegend) ex-
pression was performed as described previously after activation with
phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (1 mM) in the
presence of monensin (10 mg/ml) (2). Before activation, the cells were
stained for surface expression of CD4 (clone RM4-5; BioLegend), CD44
(clone IM7; BioLegend), CCR9 (clone 242504; R&D Systems), a4b7
(clone DATK32; BioLegend), and/or aEb7 (Clone 2E7; BioLegend).
Stained cells were analyzed using a BD Canto II (BD Biosciences).
Th17 cells generated in vitro (1 3 107cells per mouse) were labeled with
CFSE or tetramethylrhodamine isothiocyanate (TRITC) and coinjected
into wild type C57BL/6 mice via a tail vein; ∼20 h later, the mice were
sacrificed and single cell suspension was prepared from selected organs
after collagenase digestion (2). The numbers of injected CFSE+or TRITC+
Th17 cells migrated into each organ were determined with flow cytometry.
The relative homing index to a organ or tissue was determined according to
%homing index for organ A ¼ ½ðNo: of CFSEþ
cells in organ AÞ=ðNo: of TRITCþ
cells in organ AÞ?=½ðNo: of CFSEþcells in inputÞ=
ðNo: of TRITCþcells in inputÞ?3100:
The percent distribution of injected Th17 cells (Fig. 1E) was calculated
based on the numbers of migrated cells into the indicated organs.
Chemotaxiswasperformed usingTranswells(CorningGlass, Corning,NY)
with 5-mm pores as described previously (27). Optimal concentrations of
CCL25 (2.5 mg/ml) and CCL19 (1 mg/ml), determined by a preliminary
titration experiment, were used.
In vivo intestinal inflammation study to assess the effector
function of Th17 cells
B6.129s7-Rag1tm1Mom/J mice were injected i.p. with 1 3 106control or
RA-induced Th17 cells. Weight change was monitored, and the mice were
sacrificed on day 30–32 postinjection when some mice became moribund.
vitamin A-sufficient and -deficient AKR/J mice were determined. Representative (A) and combined (B) data of three independent experiments are shown.
*Significant differences between RA and control Th17 cells.
Th17 cells are decreased specifically in the small intestine of vitamin A-deficient animals. The frequencies of Th17 cells in various organs of
including the lamina propria and GALTs. A, Expression of CCR9 and a4b7
i.v.,and themigrationofTh17cells tothe indicatedorgans weredetermined
20 h after cell injection. C, The localization of the injected Th17 cells in the
indicated tissues was determined with a confocal microscope (original
magnification 3200). The experiments were performed three times, and
combined data (B) or representative data (C) are shown. *Significant
RA Th17 cells use Itgb7 to migrate to the whole intestine
lamina propria. A, Expression of CCR9 and a4b7 by wild type and CCR9
(2/2) RA Th17 cells. B, The Th17 cells were injected i.v. and the short-
term (20 h) migration of Th17 cells was determined. C, The localization of
the Th17 cells was determined with confocal microscopy (original mag-
nification 3200). Combined data (B) or representative data (C) of three
independent experiments are shown. *Significant changes.
RA Th17 cells use CCR9 to migrate to the small intestinal
The Journal of Immunology5521
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Intestinal inflammation in Rag1-deficient mice was scored as previously
described with some modification (27). We scored the degree of in-
flammation and the degree of mucosal hyperplasia and loss of villi on
a scale of 0–4. The two scores were combined to obtain the final in-
flammation index. The histologic images were obtained with a bright-field
Leica (Leica Microsystems, Deerfield, IL) microscope equipped with
a color camera at 3200 magnification.
Confocal analysis to determine localization of injected Th17 cells
Indicated tissues (spleen, peripheral lymph nodes, mesenteric lymph nodes,
and Peyer’s patches [PPs]) were harvested from euthanized mice that were
previously injected with cells stained with TRITIC and CFSE, and frozen
in Tissue-Tek (Sakura, Torrance, CA). Six-micrometer frozen tissue sec-
tions were fixed in acetone and stained with anti–CD3-APC. A Zeiss LSM
710 confocal microscope system (Zeiss, Thornwood, NY) was used to
image the cells within the tissues.
Student paired and unpaired two-tailed t tests were used to compare the
significance of differences in means of two groups of related or unrelated
data. p # 0.05 was considered significant.
Naive T cells differentiate into CCR9+a4b7+Th17 cells at
physiologic concentrations of RA
RA (i.e., All-trans-RA or At-RA) is the major biologically active
metabolite of vitamin A. To determine the role of RA in induction
of gut homing Th17 cells, we activated naive T cells in a Th17
cell-inducing condition in the presence of increasing concen-
trations of RA. The induction of Th17 cells was reduced but still
occurred at significant levels in the presence of RA lower than 30
nM (Fig. 1A). Severe reduction occurred only at high concen-
trations of RA ($100 nM). An interesting feature of these RA
Th17 cells was the expression of CCR9 and a4b7 (Fig. 1B);
however, they poorly expressed CD103 (or aEb7), another mu-
cosal homing receptor (Supplemental Fig. 3). Compared with
CCR9, a4b7 was more readily expressed by the Th17 cells at low
(∼1 nM) RA concentrations (Fig. 1B). a4b7 was induced at
a medium level, even in the absence of exogenous RA, perhaps in
response to the basal level of RA present in the serum. The in-
duction of CCR9 and a4b7 on Th17 cells by RA was prompt,
occurring after one to two cell divisions following T cell activa-
tion (Supplemental Fig. 1). Only RA Th17 cells were able to
migrate to the CCR9 ligand CCL25 (Fig. 1C). Expression of a4b7
induced by RA was relatively more stable than that of CCR9 on
Th17 cells upon subsequent T cell activation in the absence of RA
(Supplemental Fig. 2). In contrast, CCR9, once induced on Th17
cells by RA, quickly disappeared upon restimulation of Th17 cells
in the absence of exogenous RA.
The RA-induced Th17 cells preferentially migrate to the small
We performed a short-term (20 h) homing assay to determine
whether RATh17 cells, induced in the presence of exogenous RA,
RA Th17 cells and control Th17 cells. RA
Th17 cells and control Th17 cells were sep-
arately injected i.p. into Rag1-deficient mice.
A, Relative weight change owing to intestinal
inflammation. B, H&E staining of the in-
testinal tissues (original magnification 3200).
C, Inflammation scores. Data obtained from
10 to 11 mice are shown. Significant differ-
ences from the no-T cell group (p) or control
Th17 cell group (pp).
The inflammatory activities of
5522RETINOIC ACID AND TISSUE TROPISM OF INTESTINAL Th17 CELLS
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have the homing potential to the intestine. Upon i.v. injection, the
Th17 cells preferentially migrated to the small intestine (Fig. 1D,
1E). RA Th17 cells, when compared with control Th17 cells
(induced in the absence of exogenous RA), were somewhat less
efficient in migration to non–small intestine tissues (Fig. 1D).
Within the small intestine, the lamina propria is the preferential
destination of RATh17 cells (Fig. 1F). In spleens, PPs, and lymph
nodes, both the injected RA and control Th17 cells were found in
the T cell area of the lymphoid tissues.
Th17 cells are decreased specifically in the small intestine of
vitamin A-deficient mice
The results shown in Fig. 1 that RA induced gut homing Th17 cells
is surprising, because it has been reported that RA suppresses the
induction of Th17 cells from naive T cells in vitro. To determine
whether vitamin A is required for the presence of Th17 cells
in vivo, we induced vitamin A deficiency in mice and examined
the frequency of Th17 cells in different organs, including the in-
testine. We found that the frequencies of Th17 cells were de-
creased in the small intestine and PPs of vitamin A-deficient mice
(Fig. 2A, 2B). However, the numbers in spleen and MLNs were
unchanged. The data suggest that vitamin A has a tissue-specific
role in population of Th17 cells, and it supports the role of RA in
generating small intestine homing Th17 cells.
Differential roles of a4b7 and CCR9 in migration of
gut-homing Th17 cells
Next, we investigated the function of a4b7 and CCR9 in the
migration of Th17 cells. First, we compared Th17 cells derived
from wild type and integrin b7 (Itgb7)-deficient mice (Fig. 3A).
Itgb7 is a subunit for both aEb7 and a4b7. Because RA Th17
cells have low expression of ItgaE (Supplemental Fig. 3), most of
the Itgb7 subunit would be paired with the a4 subunit. We per-
formed a 20 h short-term homing assay to compare wild type and
Itgb7-deficient RATh17 cells in migration to various organs. The
migration of Itgb7-deficient Th17 cells to the intestine including
MLN, PP, small intestine, colon, and cecum was defective (Fig.
3B). Instead, the migration of these Th17 cells to other nongut
tissue sites, such as peripheral lymph nodes (313% 6 52), spleen
(235% 6 9.5), liver (166% 6 48), and marrow (206% 6 78), was
generally enhanced. The Itgb7-deficient Th17 cells failed to enter
MLNs, PPs, and the lamina propria of the intestine (Fig. 3C).
When CCR9-deficient RATh17 cells were compared with wild
type Th17 cells (Fig. 4A), they were defective in migration only to
the inflammatory activity of RA Th17 cells.
RATh17 cells that are sufficient or deficient
in expression of CCR9 or Itgb7 were sepa-
rately injected i.p. into Rag1-deficient mice
to induce inflammation. A, Weight change
owing to intestinal inflammation (n = 10–
11). B, H&E staining of the intestinal tissues
(original magnification 3200). C, Histo-
logic scores (n = 10–11). *Significant dif-
ferences from the wild type RA Th17 cell
Roles of CCR9 and Itgb7 for
The Journal of Immunology5523
by guest on June 18, 2013
the small intestine lamina propria (Fig. 4B, 4C). Although not sta-
to other mucosal tissues, such as MLNs (163% 6 34), the colon
(180%656), PPs(155%630), andthe peritoneal cavity(148%6
16), was increased (Fig. 4B). Overall, compared with a4b7, CCR9
had a more specific role in the migration of Th17 cells to the small
intestine lamina propria.
RA regulates the gut tissue tropism of Th17 cells in
We hypothesized that RATh17 cells and control Th17 cells would
induce inflammation in different locations of the intestine because
these two T cell populations have different tissue tropisms. To test
this hypothesis, the RA Th17 cells and control Th17 cells were
separately injected i.p. into Rag1-deficient mice. We found that RA
Th17 cellsinduced greaterweight losscomparedwithcontrolTh17
cells that were induced in the absence of RA (Fig. 5A). Mice
injected with control Th17 cells had more severe inflammation in
the distal colon than in the small intestine. In contrast, mice in-
jected with RATh17 cells had the most severe inflammation in the
small intestine (Fig. 5B, 5C). The inflammation in the small in-
testine was accompanied by mucosa/crypt hyperplasia and loss of
villi (Fig. 5B, 5C). The inflammation consisted primarily of in-
filtration by lymphocytes and macrophages with fewer neutrophils
and occasional eosinophils. In support of the tissue tropism of RA
Th17 cells, many more Th17 cells were found in the Rag1-
deficient mice injected with RATh17 cells compared with control
Th17 cells, even ∼30 d after the injection of the Th17 cells
(Supplemental Fig. 4). In contrast, there was no statistically sig-
nificant difference in numbers of Th1 and T regulatory cells in the
small intestine. Overall, the RATh17 cells and control Th17 cells
induce inflammation largely in distinct segments of the intestine.
The higher inflammatory activity of RATh17 cells could be due
to their differences in expression of trafficking receptors and mi-
gration ability. Alternatively, this is due to other nonmigratory
features of the cells. We compared the inflammatory activities of
wild type and CCR9 or Itgb7-deficient RA Th17 cells (Fig. 6A).
We found that both CCR9 (2/2) RA Th17 cells and Itgb7 (2/2)
RA Th17 cells had decreased inflammatory activities compared
with wild type RA Th17 cells. Particularly, the Itgb7 deficiency
completely blocked the inflammatory activity of RATh17 cells in
all segments of the intestine (Fig. 6B). In contrast, CCR9 de-
ficiency led to blockade of inflammation largely in the small in-
testine (Fig. 6C).
The decreased inflammation by CCR9 (2/2) RATh17 cells and
Itgb7 (2/2) RATh17 cells was consistent with decreased numbers
of Th17 and Th1 cells in the small intestine (Fig. 7). Moreover, the
number of Th1 cells was decreased in the large intestine of the
mice injected with Itgb7 (2/2) RA Th17 cells. FoxP3+T cells
were increased in the spleen and large intestine of the mice in-
jected with CCR9 (2/2) or Itgb7 (2/2) Th17 cells. These results
show that the two trafficking receptors that are induced by RA—
CCR9 and a4b7—have essential functions in induction of the
intestinal inflammation initiated by RA Th17 cells.
We report the presence of Th17 cell subpopulations with distinct
in vivo tissue tropism that is regulated by the RA signal. The tissue
tropism of these Th17 cells is determined by differential expression
of gut homing receptors. Strikingly, the Th17 cell subpopulations
with different tissue tropism induced inflammation in different
parts of the intestine. We believe that this is the first demonstration
of the presence of Th17 cell subsets with inflammatory activities in
distinct compartments of the intestine.
What concentration of RA is really available for T cells un-
dergoing Ag activation in vivo? The RA concentration in normal
human blood plasma is ∼4.9 ng/ml (28). Comparably, ∼2 ng/ml of
RA was detected in rat serum. It is estimated that 50–75% of the
RA in serum or plasma is At-RA. Therefore, the concentration of
At-RA in normal blood plasma is estimated to be ∼5 nM. The
balance between RA-synthesizing retinaldehyde dehydrogenases
and RA-inactivating cytochrome P450RAI (CYP26) determines
the availability of RA in a given tissue microenvironment. The
expression patterns of CYP26 and RA-synthesizing retinaldehyde
dehydrogenase type 2 are largely complementary in developing
embryos (29–31), and it is expected that a similar pattern of
complementary expression of the two enzymes would occur in
adults. Based on the sensitivity of the expression of CCR9 and
a4b7 in response to RA, it is estimated that the intestinal Ag-
priming environment would have at least 2–3 nM and potentially
higher levels of active RA (,30 nM). The concentration of RA in
peripheral lymph nodes, where CCR9 is not induced, is expected
to be ,1 nM. We estimate that RA levels between 0.1 and 30 nM
are within the physiologic range. RA concentrations higher than
this range could be found in vivo but the need for that high RA
signal is unclear.
A most characteristic feature of the Th17 cells induced at
physiologic levels (10–20 nM) of RA is the expression of both
CCR9 and a4b7. These two trafficking receptors are somewhat
different from each other in requirement for RA for persistent
expression. a4b7 initially induced by RA persists, even after
subsequent Ag priming in the absence of exogenous RA. In
contrast, expression of CCR9 on Th17 cells is transient requiring
continued presence of RA. Moreover, CCR9 is not induced well in
response to RA on the Th17 cells that were previously activated in
the presence of suboptimal concentrations of RA. We found that
a4b7 is a general homing receptor of Th17 cells for the intestine
and associated lymphoid tissues, whereas CCR9 is a receptor
more specific for the small intestine. This finding suggests that at
least several populations of Th17 cells (CCR9+a4b7+, CCR92
a4b7+, and CCR92a4b72) with different homing behaviors can
cells and effector T cell balance in the intestine. RA Th17 cells that are
sufficient or deficient in expression of CCR9 or Itgb7 were injected i.p.
into Rag1-deficient mice, and absolute numbers of Th1 and Th17 cells in
the spleen, small intestine, and large intestine were determined ∼30 d later.
Absolute numbers of the T cells per organ detected by the method are
shown. Combined data with averages and SEM are shown (n = 10–12 per
group). *Significant differences from the wild type RA Th17 cell group.
Roles of CCR9 and Itgb7 in long term population of Th17
5524RETINOIC ACID AND TISSUE TROPISM OF INTESTINAL Th17 CELLS
by guest on June 18, 2013
be generated in different sites of the intestine, depending on the
RA availability during repetitive priming processes.
The potent activity of retinoid-induced Th17 cells in inducing
small intestine inflammation is notable and is associated with the
expression of a4b7 and CCR9. a4b7 is widely expressed by in-
testinal lymphocytes and serves as the major homing receptor (32,
33). a4b7 is an adhesion molecule for MAdCAM-1 (32, 34) and
can guide the Th17 cells to the entire gut system. It appears that
even basal levels of a4b7, induced in the presence of low (serum)
levels of RA, are required for migration of Th17 cells into the
intestine and associated lymphoid tissues. Thus, higher expression
of a4b7, induced at optimal levels of RA over the physiologic
range, confers Th17 cells with the enhanced ability to stay within
the intestine and to induce inflammation. CCR9 is expressed more
specifically by the T cells in the small intestine and acts as
a homing receptor that is specific for this tissue (35–38). CCR9
can guide the Th17 cells to the small intestine lamina propria
compartment close to epithelial cells where CCL25 is expressed
(35, 39, 40), and therefore would cause the more focused migra-
tion and inflammation in the small intestine. Consistently in our
study, Itgb7 deficiency had a broader effect on Th17 cell migra-
tion to the intestine, whereas CCR9 deficiency had a specific effect
on their homing to the small intestine. In line with the differential
roles of these trafficking receptors in determining the tissue tro-
pism of Th17 cells, Itgb7 deficiency had greater impact than
CCR9 deficiency on the inflammatory activity of the retinoid-in-
duced Th17 cells. This finding is supported by the fact that,
without proper expression of Itgb7 or a4b7, Th17 cells fail to
migrate to any site of the entire intestinal system, including the
small intestine, despite their normal expression of CCR9. This
finding is in line with the established roles of these trafficking
receptors for other T cells (32, 33, 35, 39, 40), and it suggests that
Th17 cells do not deviate from the general trafficking behavior of
gut T cells. Some studies have reported that IL-22 has a protective
role in intestinal inflammation (12, 41), whereas others reported
proinflammatory roles of IL-22 in intestinal inflammation (42, 43).
We would like to point out that there was no significant difference
in expression of IL-22 by control and RA Th17 cells (Supple-
mental Fig. 5).
Because of the previously reported in vitro phenomenon that
induction of Th17 cells is suppressed by RA (19, 22, 23), it has
been assumed that vitamin A antagonized the population of Th17
cells in the intestine. Our finding with vitamin A-deficient mice
indicates that the RA signal is actually important for the presence
of Th17 cells in the small intestine. A similar decrease of small
intestine Th17 cells in vitamin A deficiency was observed in
a mouse model of chronic intestinal inflammation (44), suggesting
that it occurs broadly in both normal and inflammatory conditions.
We do not rule out the possibility that induction of Th17 cells
could be somewhat suppressed by the RA in the small intestine
in vivo. However, the increase of gut homing receptor-expressing
Th17 cells would compensate for this small decrease caused by
the presence of RA. Thus, there would be an overall increase in
the number of gut Th17 cells in the intestine in response to RA.
Our results provide useful targets of intervention in regulation of
the inflammatory activities of Th17 cells in the intestine. It is
possible to regulate the inflammatory activity of Th17 cells in the
small versus other compartments of the intestine by selective
blocking of CCR9 or Itgb7. This projection is well supported by
therapies approved by the U.S. Food and Drug Administration and
other proposed therapies targeting a4 or a4b7 integrin. For ex-
ample, blocking of the Itga4 using mAbs to integrin a4 is
a treatment for Crohn’s disease approved by the U.S. Food and
Drug Administration (45). It was also shown to be effective in the
amelioration of colitis in animal models (45–47). Whereas this
therapy targets all a4 integrins including a4b7 and a4b1, mAbs
more specifically blocking Itgb7 or MAdCAM-1 were also ef-
fective in inhibiting colitis in animal models (48, 49). We propose
that these reagents can act on the a4 integrins of Th17 cells and
can therefore block their migration and inflammatory activities in
the intestine. If the inflammatory activity of Th17 cells is localized
to the small intestine, blocking of CCR9 would be more specific in
treating the localized inflammation without the potential side ef-
fects of broad immune-suppression in the body.
We thank J.S. Chang, J.H. Lee, and Jeongho Park (Purdue University) for
The authors have no financial conflicts of interest.
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