Physiol Rev 95: 125–148, 2015
doi:10.1152/physrev.00032.2013
LEUKOCYTE HOMING, FATE, AND FUNCTION ARE
CONTROLLED BY RETINOIC ACID
Yanxia Guo, Chrysothemis Brown, Carla Ortiz, and Randolph J. Noelle
Department of Microbiology and Immunology, Dartmouth Medical School, Norris Cotton Cancer Center,
Lebanon, New Hampshire; and Medical Research Council Centre of Transplantation, Guy’s Hospital, King’s
College London, King’s Health Partners, London, United Kingdom
Guo Y, Brown C, Ortiz C, Noelle RJ. Leukocyte Homing, Fate, and Function Are Controlled
by Retinoic Acid. Physiol Rev 95: 125–148, 2015; doi:10.1152/physrev.00032.2013.—
Although vitamin A was recognized as an “anti-infective vitamin” over 90 years ago, the
mechanism of how vitamin A regulates immunity is only beginning to be understood.
Early studies which focused on the immune responses in vitamin A-deficient (VAD)
animals clearly demonstrated compromised immunity and consequently increased susceptibility to
infectious disease. The active form of vitamin A, retinoic acid (RA), has been shown to have a
profound impact on the homing and differentiation of leukocytes. Both pharmacological and genetic
approaches have been applied to the understanding of how RA regulates the development and
differentiation of various immune cell subsets, and how RA influences the development of immunity
versus tolerance. These studies clearly show that RA profoundly impacts on cell- and humoralmediated immunity. In this review, the early findings on the complex relationship between VAD and
immunity are discussed as well as vitamin A metabolism and signaling within hematopoietic cells.
Particular attention is focused on how RA impacts on T-cell lineage commitment and plasticity in
various diseases.
L
I.
II.
III.
IV.
V.
VI.
VII.
INTRODUCTION
VITAMIN A DEFICIENCY AND...
VITAMIN A METABOLISM AND RA...
RA SIGNALING PATHWAY
RA IN REGULATION OF T CELLS
RA REGULATION OF T-CELL IMMUNITY...
CONCLUDING REMARKS
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I. INTRODUCTION
Nearly a century ago, vitamins were recognized as essential
nutrients for human health. Among various vitamins, vitamin A was found to be essential for embryogenesis (215)
and host defense against various microbial infections (209).
Developmental biologists have extensively studied the active form of vitamin A, retinoic acid (RA), and have mapped
how grandients of RA control the most exquisite processes
during embryogenesis. Given the known correlation between vitamin A deficiency (VAD) and increased susceptibility to infectious diseases (ID) among preschool children
in developing countries (20, 49), immunologists have focused on the understanding of how vitamin A, through RA,
controls immunity. Initial clues into the molecular mechanisms of the “anti-infective” impact of vitamin A were provided in 2004, when Iwata et al. (84) demonstrated that RA
imprints the homing of leukocytes to the gut through the
upregulation of ␣4␤7and chemokine (C-C motif) receptor 9
(CCR9). Our own studies (11, 140) and those of others (31,
139, 181) demonstrated that RA could enhance T-cell com-
mitment and Treg differentiation. These seminal findings
focused the immunological community on understanding
the role of RA in the regulation of gut immunity and tolerance. The use of small molecule inhibitors or agonists of RA
signaling as well as genetic-engineered mouse models has
expanded our knowledge of how RA controls immune responses besides its known function in controlling gut homing. Numerous studies have elucidated the essential role of
RA in modulating immune cell “lineage commitment” in
various disease models in a context-dependent manner. RA
is now appreciated as a pivotal mediator in promoting tolerance or immunity depending on what type of cell is acted
upon. In this review, we focus on the known mechanism of
how RA is synthesized by various antigen-presenting cells
and how this is translated into signals that govern leukocyte
lineage commitment.
II. VITAMIN A DEFICIENCY AND
IMMUNITY
Vitamins by definition are organic compounds that cannot
be synthesized by mammals and thus have to be obtained
from food sources (52). The recognition that vitamin A
plays an important role in immunity dates back to the
1920s when Drs. Green and Mellaby observed their colony
of dogs on a VAD diet succumbed to an infection that
ravaged through the animal facility (127). Since that time,
the idea that vitamin A was an “anti-infective vitamin”
instead of “growth-producing vitamin” (62) grew in recog-
0031-9333/15 Copyright © 2015 the American Physiological Society
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GUO ET AL.
nition. Following this study, a small clinical trial was initiated in women with septicemia, in which only 2 out of 24
recovered without vitamin A treatment, and 5 out of 5
recovered with vitamin A treatment (128). An even larger
clinical trial of puerperal sepsis reinforced the positive correlation between vitamin A treatment and clinical outcome
by showing a statistical improvement in a wide range of ID
of those on vitamin A-rich diets (63). At least 30 uncontrolled clinical trials led by Drs. Mellanby and Green demonstrated that vitamin A supplementation, in the form of
cod-liver oil, reduced the morbidity and mortality caused by
respiratory inflammation, measles, puerperal sepsis, and
other ID (as reviewed in Ref. 165). A litany of studies have
focused on the analysis of VAD and immunity, with the
increasing knowledge of immunity and better-controlled
experiments than in the early days. VAD animals are characteristic of a wide range of immunological changes, including pathological disintegrity in mucosal area (2), decreased
antibody (Ab) response, changes in lymphocyte populations, and T- and B-cell dysfunction, which can be restored
by treatment with retinol, a derivative of vitamin A (154).
The importance of vitamin A in human disease is highlighted further by the increased burden of ID in VAD children and adults. Presently, VAD is still a common health
problem in some developing countries and a leading cause
of childhood morbidity and mortality. An estimated 250
million children under the age of 5 yr have VAD (190),
defined as serum retinol ⬍0.7 ␮M (37), and a further 120
million are estimated to have significant VAD in the absence
of clinical signs such as xerophthalmia. Malnourished children have increased susceptibility to diarrhea due to ID (49)
and an increased risk of acute respiratory infections (20)
and measles (54, 122). Some clinical data also indicate a
correlation between VAD and the disease progression
(166), mortality (166), and maternal-infant disease transfer
(64, 167). Infection also leads to enhanced vitamin A depletion (21). Since the first randomized and controlled trial in
the 1980s, a number of clinical trials in children from endemic areas have demonstrated a dramatic reduction in
severity of diarrheal disease and measles morbidity with
vitamin A supplementation (78, 81, 172). In addition, supplementation has led to a lower incidence of respiratory
tract infections in some studies (9). Of note, high-dose vitamin A supplementation even reduced mortality and morbidity in children without clinical evidence of VAD (32, 78).
Based on these clinical outcomes, the World Health Organization recommends that preschool children in developing
countries receive high-dose vitamin A supplementation every 4 – 6 mo, often administered at the time of vaccination
for clinical convenience.
In addition to the role of vitamin A sufficiency to efficiently
fight against infectious diseases, evidence also suggests that
vtiamin A supplementation can improve vaccine efficacy
(92). The study by Sudfeld et al. (180) showed that at least
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two doses of 200,000 IU (60 mg retinol equivalent) for
children ⱖ1 yr of age and 100,000 IU (30 mg retinol equivalent) for infants reduced measles mortality by 62%. In the
study, vitamin A is administrated orally in an oil-based
preparation of retinyl palmitate or retinyl acetate. However, the relationship of vitamin A supplementation and
mechanistic impact on immunity is not always apparent.
Chicks fed on a high-vitamin A diet had a significantly
lower primary IgG and IgM Ab response to keyhole limpet
hemocyanin (KLH), and also a lower secondary IgG response. In addition, administering retinyl palmitate did not
change the Ab response in low-, regular-, or high-vitamin A
diet-fed chicks (108). VAD diminished and high-level vitamin diet enhanced the development of experimental asthma
in mice (162). Furthermore, vitamin A supplementation
proved unpredictable in some clinical trials. In a clinical
trial including 471 at-birth children, vitamin A supplementation (50,000 IU retinyl palmitate in vegetable oil) was
associated with lower tumor necrosis factor (TNF)-␣ and
interleukin (IL)-10 production in girls without and boys
with diphtheria-tetanus-pertussis vaccination, indicating a
complicated interaction between vitamin A sufficiency, gender, and vaccination (88).
As discussed above, the early epidemiological data and effectiveness of vitamin A supplementation to boost immunity shed light on a key role of vitamin A for development of
host defense and opened the door to a wealth of research
into the mechanisms through which vitamin A regulates
immune responses. On the other hand, the insignificant improvement by vitamin A or RA supplementation in some
diseases makes it clear that vitamin A effects are not always
predictable.
The importance of vitamin A to the ontogenic development
of immune system was recognized in 1930s, when VAD
children were found to have atrophic thymi, spleens, and
lymphoid tissues (15). Numerous studies in mice have demonstrated that vitamin A causes this impact on immune
system through various immune cell components, such as
NK cells (39), T cells (110), and B cells (16). Recently, the
importance of vitamin A in development of immune system
was further understood at the molecular level by van de
Pavert et al. (197). In this study, the authors established that
the secondary lymphoid organ formation during embryogenesis depends on hematopoietic cell-autonomous RA signaling in utero, by control of a subset of type 3 innate
lymphoid cells (ILC3) differentiation. Therefore, maternal
vitamin A intake controls the efficiency of immune response
in the adult offspring. It further illustrated the essential role
of vitamin A in the development of efficient immune system.
III. VITAMIN A METABOLISM AND RA
SYNTHESIS
Both genetic and pharmacological approaches have been
applied to elucidate the pathway of metabolizing vitamin A
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RETINOIC ACID AND IMMUNITY
to RA. As retinoids (all vitamin A derivatives) cannot be
synthesized de novo, the only source is diet derived. Earlier
studies have illustrated the major proteins and enzymes involved in retinol storage are in the liver and the uptake of
vitamin A is in the intestine (reviewed in Ref. 33). In mammals, retinol is the major circulating retinoid. Retinol is
typically bound to retinol-binding protein 4 (RBP4) and
transthyretin (TTR), and taken up by target tissues (10).
Previous studies demonstrated that retinol-RBP-TTR complex (holo-RBP) uptake was facilitated by STRA6 (stimulated by retinoic acid gene 6) in various tissues (93). However, recent studies in stra6-null mice illustrated that
STRA6 facilitates retinol uptake in the eye but couples holoRBP to cell signaling in other tissues (12). Two sequential
reactions are then carried out for the conversion of retinol
to RA. First, retinol is converted by cytosolic alcohol dehydrogenase (ADHs, including ADH1, ADH5, and ADH7)
(133) and microsomal retinol dehydrogenase (RDH) (158)
to retinaldehyde. Adh1⫺/⫺ mice are more sensitive to vitamin A embryotoxicity (134), but reveal no obvious phenotype when maintained on vitamin A-sufficient diet (38). In
contrast, Rdh10 loss-of-function is lethal between embryonic day 10.5 (E10.5) and E14.5 and exhibits abnormalities
characteristic of RA deficiency (158). These studies indicated that ADHs may control the removal of excessive retinol, whereas RDHs oxidize retinol to retinaldehyde (also
called retinal). Second, retinaldehyde is irreversibly converted to all-trans-retinoic acid (ATRA) by three retinaldehyde dehydrogenases, RALDH1 (65), RALDH2 (43, 144)
and RALDH3 (182) (encoded by Raldh1–3, respectively),
in mice. In this article, RA refers to ATRA unless specificed
otherwise. Of note, RALDH2 can also catalyze the dehydrogenation of other aldehydes, such as octanal and decanal, even if less efficiently compared with retinaldehyde
(208).
Even though vitamin A has long been known to be essential
for a competent immune system, the underlying mechanisms whereby RA regulates immunity are still incompletely understood. Substantial efforts have focused on the
metabolism and synthesis of RA by various APCs for the
homeostatic regulation of immune migration and differentiation of various hematopoietic compartments.
A. RA Synthesis in the Gut
While ADHs are widely and constitutively expressed by
multiple cell types, RALDH expression is dynamically regulated in a temporally and spatially restricted manner. Expression of RALDH is viewed as a key enzyme defining the
ability of specific cell types or tissues to produce RA (43, 65,
144, 182). The discovery that RA induces the migration of
leukocytes to the gut (84, 137, 223) has focused much of the
attention of RA synthesis to mucosal sites. As such, researchers have begun to elucidate the factors and cells that
govern the expression of RALDHs and RA synthesis in
immune relevant cells in the gut and in the periphery. In situ
hybridization and immunocytochemical analysis have confirmed RALDH1 expression in epithelial cells of mucosa,
including small and large intestines (55). In the small intestine, epithelial cells express Raldh1 and produce RA constitutively by taking retinol from food sources (14, 55, 104,
192). Further studies found that monocyte-derived dendritic cells (MoDC) differentiated with RA or the supernatant of human intestinal epithelial cells acquire the ability to
produce RA and induce regulatory T cell (Treg) differentiation in the gut (80). It has also been shown that direct
contact between dendritic cells (DC) and intraepithelial
lymphocytes (IELs) endowed the former to express Raldh2
and produce RA (47, 79). Many studies have suggested that
CD103⫹ DCs in the small intestine are responsible for the
high level of RA in mesenteric lymph node (MLN)
(RALDH2), Peyer’s Patch (PP) (RALDH1), and lamina propria (LP) (RALDH2) of the small intestine (84). Recent
studies have shown that not only are there more CD103⫹
DCs in murine small intestine lamina propria (SI-LP) and
draining MLN than in other lymphoid tissues, but CD103⫹
DCs in SI-LP and MLN showed higher Raldh2 expression
and activity (132).
Our recent study demonstrated that both the expression
and enzymatic activity of RALDH1 and RALDH2 in donor
DCs and macrophages of MLN and LP lymphocytes were
elevated in graft-versus-host-disease (GVHD) mice (5). The
increase of RA synthesis in active GVHD mice is responsible
for donor T cells differentiation into pathogenic Th1/Th17
cells and their migration to the instestine for disease progression.
Another study showed that RALDH2 is also expressed in
MLN stromal cells, consistent with their ability to imprint
proliferating T cells with ␣4␤7 and CCR9. This study suggested that RA-producing stromal cells and DCs in MLN
warrant the gut-tropism of activated T cells in the musoca
area (131). In one elegant study of implanting MLN and
peripheral LN fragments into the mesenteric region of host
rat and mouse, it was established that stromal cells in MLN
but not in peripheral LN provided a unique microenvironment allowing for the acquisition of a gut-tropism phenotype of T and B cells during antigen-induced activation (3,
72). In addition to gut mucosa, CD103⫹ DCs was also
found in cutaneous and lung-draining LN, suggesting an
important role in controlling T cell homing in those sites
(67). A recent study demonstrated that lung CD103⫹ and
CD24⫹CD11b⫹DCs express RALDH activity and can induce gut-homing receptors ␣4␤7 and CCR9 (156). Even
though no specific RALDH was identified, T cells primed by
lung DCs via intranasal immunization efficiently induced
migration to the gut and control enteric challenge of highly
pathogenic Salmonella. While these correlations of expression and function within the CD103⫹ DC subset are compelling, it remains to be determined whether RA synthesis
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by these cells are causally imperative for the function of RA
in the gut.
T-cell recruitment into the intestine and ameliorates the
disease progression.
B. RA Synthesis in the Periphery
C. Regulation of RA Synthesis in the
Immune System
Various peripheral DC subsets acquired RA-producing potential in both allogenic skin transplantation (151) and melanoma model (69). As such, our studies have established
that RA is a critical immune mediator outside the gut, in
sustaining and controlling leukocyte differentiation in the
periphery. RA production by DCs in peripheral tissues and
lymphoid organs is consistent with the corresponding role
of RA in regulating adaptive immunity in periphery. Furthermore, CD8␣⫹CD8␤⫺ and CD8␣⫹CD8␤⫹ but not
CD8␣⫺CD8␤⫺ plasmacytoid DCs (pDC) were able to produce RA, and induce the conversion of naive CD4⫹ T cells
to Tregs in combination with transforming growth factor
(TGF)-␤ in vitro and in vivo during lung inflammationmediated airway hypersensitivity (109).
As mentioned in section II, the intake of retinoids by the
mother determines the development of secondary lymphoid organs during embryogenesis through the regulation of lymphoid tissue inducer (LTi) cells and
CD3 ⫺ IL7R ␣ ⫹ ␣ 4 ␤ 7 ⫹ ID2 ⫹ c-Kit ⫹ CD11c ⫺ CD4 ⫺ innate
lymphoid cells (ILCs) as early as E10.5 (197). RALDH2 is
responsible for RA synthesis until E8.5 (130, 144, 145), and
thereafter RALDH1 and RALDH3 control RA synthesis
(46, 125). The authors proposed that RA produced in utero
operates in a cell-autonomous fashion to regulate retinoic
acid receptor-related orphan receptor gamma (ROR␥T), a
key transcription factor for the development of LTi cells. It
remains unknown which RALDH is required for RA production and provision for LTi cell differentiation and secondary lymphoid organs generation.
No studies have yet reported RALDHs expression in T cells
or RA synthesis by activated T cells in vitro or in vivo. The
exclusive synthesis of RA by various APC subsets and impact on T cell differentiation raised the question of whether
RA, as a lipophilic molecule, permissively diffuses across
cell membranes of DCs and T cells during antigen presentation and thus determines the phenotype of corresponding
T cells. Most likely, RA-producing DCs (or other APCs), in
response to different stimuli, can generate an RA-rich microenvironment, which is also rich in stimuli-specific cytokines/chemokines, and determine the T-cell lineage commitment. Lineage-specific deletion of Raldh(s) will facilitate the
understanding of the role of each RALDH in DCs or macrophages in the regard of determining T-cell differentiation
and migration. Such studies may lead to the therapeutic
inhibition of RALDH activity in specific cell subsets for
control of disease progression. For instance, Crohn’s disease and ulcerative colitis are driven by enhanced recruitment of effector macrophages, neutrophils, and effector T
cells (159). Thus RALDH inhibition may block effector
128
Interest in understanding what inflammatory mediators
control RA synthesis by the immune system has been paramount. The prominent targets for regulation involve the
transcriptional regulation of each of the RALDHs as well as
the expression of the RA catabolic pathways, involving
mainly cytochrome P-450, CYP26 enzymes (CYP26A1C1), and possibly retinal reductases (89). The following
section focuses on our current understanding of the molecules involved in the expression and activity of RALDH1–3
and CYP26 in different immune cells.
1. Granulocyte-macrophage colony stimulating factor
and IL-4
Granulocyte-macrophage colony stimulating factor (GMCSF) and IL-4 have been shown to synergistically induce
Raldh2 expression in both bone marrow-derived DCs
(BMDC) and splenic DCs, thus enabling them to produce
RA (6). Even though 1 ␮M ATRA minimally induced
Raldh2 expression in BMDC or splenic DC, it moderately
enhanced GM-CSF and/or IL-4-induced Raldh2 expression
on BMDCs. The authors proposed a positive feedback loop
of RA production in the intestine. That is, GM-CSF produced by CD11c⫺F4/80⫹ macrophages in the intestinal tissues induces Raldh2 expression and RA production by
DCs. Raldh2⫹ DCs then provide RA to macrophages and
induce further GM-CSF production. A recent study further
elucidated that RA controls the generation of gut-tropic
migratory DC precursors in the bone marrow (223). Therefore, high RA in the intestinal microenvironment is maintained through the generation of gut-homing DC precursors
in bone marrow and further enhancement of RA production by GM-CSF in the intestine. Consistent with the finding that GM-CSF and IL-4 induced Raldh2 expression on
BMDCs, IL-4 was shown to induce Raldh2 expression in
MLN-DC (50).
2. TLR signaling
The RA-producing ability of CD103⫹ DCs in the gut mucosa is dependent on MyD88 signaling, in particular TLR2mediated signaling (48, 206). TLR2 signaling also stimulated RALDH2 and IL-10 expression on splenic DCs, thus
inducing Treg development but suppressing Th1/Th17 cell
differentiation (119). Another study showed that TLR5 ligand, flagellin, specifically stimulated CD103⫹ LP-DCs to
produce RA (194). The MyD88 and TLR signaling-dependent RALDH2 expression indicate the potential ability of
both commensals and pathogens to induce RA synthesis by
gut-associated DCs. Consistent with this, infection with
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RETINOIC ACID AND IMMUNITY
Candida albicans was also shown to induce Raldh2 expression in splenic DCs (119). Surprisingly, both MyD88⫺/⫺
Trif Lps2/Lps2 mice (that lack central adaptors used by all
TLRs and thus lack all signals via TLR) and wild-type
C57BL/6 mice kept under specific pathogen-free or germfree conditions showed moderately diminished RALDH activity in MLN DCs, indicating a redundant or distinctive
pathway regulating the steady-state RALDH2 expression
on MLN DCs (67). Thus regulation of RALDH2 in steadystate DCs may involve distinct pathways from RALDH2
expression induced by inflammation. More recent studies
found that infection with the helminth Shistosoma mansoni, which provokes Th2 response, led to massive
RALDH2 upregulation within alternatively activated macrophages (17). Interestingly, human mast cell-derived IL-3
can induce RALDH2 expression in basophils, thus inducing
Th2 response in allergic diseases (174). However, no
RALDHs induction was observed following infection with
lymphocytic choriomeningitis virus (LCMV), highlighting
the pathogen-specific induction of RA synthesis (17).
3. 4-1BB and prostaglandin E2
A recent study found that 4-1BB⫹CD103⫹ DCs in MLN
displayed higher RALDH activity than 4-1BB⫺CD103⫹
DCs. 4-1BB stimulation allowed TLR2-pretreated splenic
DCs to be more potent Treg inducers. The essential role of
4-1BB for steatdy-state RALDH2 expression on MLN-DCs
was confirmed further by weak RALDH2 expression and
Treg-promoting activity in MLN-DCs of 4-1BB-deficient
mice (105). As it is essential for the gut-associated lymphoid
organs to fight against foreign pathogens while maintaining
tolerance towards food antigens, there needs to be negative
regulation of RA synthesis by MLN DCs, thus eliciting
anti-inflammatory response. Prostaglandin E2 was indeed
shown to block GM-CSF-induced RALDH2 expression in
both murine and human DCs, providing a potential mechanism for suppression of RA synthesis by DCs at steady
state, since prostaglandin E2 is produced by stromal cells as
wells as DCs (178). The coordinated RA synthesis by proinflammatory and anti-inflammatory signals highlights the
essential role of RA playing in maintaining the balance between tolerance and immunity.
4. Wnt signaling
␤-Catenin-stimulated Wnt signaling was upregulated in
LPDCs compared with splenic DCs. Further studies illustrated that ␤-catenin is required for steady-state Raldh1 and
Raldh2 expression at mRNA level in intestinal DCs and
macrophages. Therefore, specific deletion of ␤-catenin in
DCs led to lower Treg but higher Th1/Th17 differentiation,
and the host is more prone to inflammatory bowel disease
(120).
We summarize the different molecules for stimulating/
maintaining RA synthesis in various cells in the gastrointestinal (GI) tract in FIGURE 1.
5. RA catabolism
To maintain RA tissue concentrations and gradients, regulation of catabolism is coupled with synthesis. A family of
Cyp26A1-C1 can transform ATRA into more polar metabolite 4-hydroxy-RA or 4-oxo-RA, which is subject to further metabolism and elimination from cells (117, 185, 212).
A recent study suggested that dehydrogenase/reductase-3
may also be involved in the catabolism of RA by reducing
the level of all-trans-retinal (89). Thus the expression and
activity of RALDHs, CYP26, and dehydrogenase/reductase-3 in combination determine the intracellular and extracellular RA levels. Cyp26A1 and Cyp26B1 show specific
activity for ATRA (211, 213), whereas Cyp26C1 shows
activity for both ATRA and 9-cis-RA (186). During early
embryogenesis, RA levels decrease in the regions where
CYP26 are expressed, thus Cyp26a1⫺/⫺ or Cyp26b1⫺/⫺
mice die in utero or immediately after birth (1, 219). The
key role of CYP26 in regulating RA concentration was also
demonstrated in immune cells. For instance, peroxisome
proliferator-activated receptor gamma (PPAR␥) agonists
induced human DCs to produce RA, which then leads to the
upregulation of CD1d, an antigen-presenting glycoprotein.
This effect was enhanced by a CYP26 inhibitor (R115866),
illustrating the essential role of CYP26 in RA catabolism in
the immune system (184).
The classical RA response element (RARE) in the Cyp26a1
promoter region and a G-rich element synergistically allow
for RA-induced Cyp26a1 expression (111, 112). In contrast, Takeuchi et al. (188) found that expression of
Cyp26b1 instead of Cyp26a1 or Cyp26c1 can be induced
by RA in naive T cells upon activation, indicating an indirect regulation of Cyp26b1 by RA in T cells. This study also
illustrated that RA-induced Cyp26b1 expression was suppressed by TGF-␤, but enhanced by proinflammatory cytokines IL-12, IL-4, and TNF-␣. These findings are consistent
with the effect of RA in 1) suppressing IL-12-mediated Th1,
2) enhancing IL-4-mediated Th2 response (83), and 3) enhancing TGF-␤-induced Treg differentiation (11, 139,
181). To better understand the role of CYP26 in regulating
regional RA level and thus differentiation or homing phenotype of immune cells, clear expression patterns of Cyp26
isoforms across hematopoietic cell lineages need to be defined. Also, conditional overexpression of specific Cyp26
isoforms in specific immune cell lineages will provide a better understanding of whether CYP26 activity determines
cellular RA level in vivo. Due to the side effects of RA
isomers in therapeutic applications, CYP26 inhibitors were
synthesized and evaluated in preclinical models (60). The
small molecules can potentially target certain specific cells
through drug-Ab conjugates (193).
IV. RA SIGNALING PATHWAY
There are three different retinoic acid receptor (RAR) subtypes: RAR␣, RAR␤, and RAR␥ (44, 222), each consisting
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Epithelial cells
CD103+
DC
CD103+
DC
RA
β-cat
Peyer’s Patch
Wnt
RA
4-1BB
ligand
GM-CSF
Mesenteric Lymph Node
PGE2
RA
CD11c–F4/80+
macrophage
RA
TLR2/5
ligand
4-1BB
CD103+
DC
RA
RA
Lamina Propria
Stromal
cells
FIGURE 1. Retinoic acid (RA) synthesis pathway in the gut. CD103⫹ DCs are major RA producers in LP,
Peyer’s patch, and MLN. Epithelial cells in the small intesine produce RA spontaneously. Intestinal epithelial
cell-derived RA enhances CD11bc⫺F4/80⫹ macrophages to produce GM-CSF, which in combination with RA
promotes CD103⫹ DCs in LP to produce more RA in SI-LP. Wnt/␤-catenin signaling is required for RA
production in LP CD103⫹ DCs. TLR2/5 ligands derived from dietary digestion products enhance RA synthesis
in CD103⫹ DCs. In contrast, PGE2 inhibits RA production. 4-1BB l is required for MLN-DCs to produce RA. In
addition, in MLN, stromal cells also produce RA, which in turn drives RA production by CD103⫹ DCs.
of at least two isoforms generated by alternative splicing
at the NH2 terminus (58, 91, 106). There are three major
domains in RARs required for its function, including
NH2-terminal domain (NTD), central DNA-binding domain (DBD), and COOH-terminal ligand binding domain (LBD) (27). RARs form heterodimer with retinoid
X receptor (RXR) to regulate target genes expression.
RAR-RXR heterodimer binds to RARE composed of two
direct repeats of a core hexameric motif, PuG (G/T) TCA
separated by 5 bp (DR5), 2 bp (DR2), or 1 bp (DR1) to
regulate target gene transcription (8). The subsequent
ligand-induced conformational changes in LBD direct the
dissociation of corepressors and association of coactivator complexes, thus inducing gene transcription (27).
Specifically, in the absence of retinoid ligand (such as
RA), RAR-RXR heterodimer recruit and maintain corepressors to the promoter region of target genes, prevent
other transcriptional machinery such as RNA polymerase binding to the promoter, and thus repress the transcription of these genes. On the other hand, however, RA
binding to the RAR-RXR heterodimer releases the corepressors from the promoter, and recruits the coactivators
and the transcription machinery so that target genes can
be transcribed. ATRA can bind to RAR, but 9-cis-RA can
bind to both RAR and RXR (57, 101, 224).
130
In addition to functioning through RAR-RXR heterodimer,
RA was also shown to be a ligand for PPARdelta. The
inconsistency between the prosurvival effect of RA and the
proapoptotic function through RAR led to the discovery of
PPARdelta as another receptor for RA signaling in specific
cell types (161). Another study also illustrated that
PPARdelta activation by RA leads to expression of the insulin-signaling gene PDK1, thus stimulating lipolysis and
reducing triglyceride content (13).
In the immune system, some direct evidence has shown that
RA can regulate target genes expression at transcription level
(188). The cross-talk between RAR␣ and nuclear factor
of activated T cells, cytoplasmic, calcineurin-dependent 1
(NFATc1)/NFATc2 proved essential for CCR9 induction in
response to transient TCR stimulation (148). In addition to
the direct impact of RA on various genes transcription, RA
was found to control microRNAs in different T-cell subsets,
which enables RA to function through posttranscriptional regulation of multiple genes (86, 187). Therefore, given the large
number of potential RAREs in the genome, RA may (in)directly regulate target genes through various mechanisms.
Various genetic models have been used to elucidate the role
of RA in controlling the immune response from whole body
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RETINOIC ACID AND IMMUNITY
to cellular and molecular level. DR5-luciferase mice have
been used to visualize immune stimulation-induced RA signaling at both the whole body and cellular levels (5, 69,
151, 183). Various studies have used transcriptional profiling to determine the target of RA signaling in T cells (76),
DCs, and other cell subsets. Conditional deletion of RARs
in specific tissues or cell subsets has also been used to reveal
the role of various RARs in controlling different aspects of
immune response. In addition, overexpression of a dominant negative RAR␣ (dnRAR␣) has been used to reveal the
function of RA signaling in controlling T-cell differentiation (4, 5, 69, 151). This serves the purpose of recapitulating VAD in the immune system. The following section will
focus on the known mechanism of RA signaling in control
of T-cell differentiation and lineage commitment.
V. RA IN REGULATION OF T CELLS
A. RA Regulates T-Cell Homing Potential
The essential role of RA in gut immunity had led researchers
to investigate the role of RA in the gut-tropism for T cells,
an essential component in gut immunity. The interaction
between intergrin ␣4␤7 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is essential for T-cell homing to intestine endothelium (19), whereas CCR9 mediates
T-cell migration to intestinal epithelial cells via interaction
with chemokine (C-C motif) ligand 25 (CCL25) (100). T
cells gain ␣4␤7 and CCR9 expression during antigen stimulation by MLN-DC or PP-DC, or ␣CD3 and ␣CD28 stimulation in the presence of RA, while downregulating E- and
P-selectin ligands simultaneously (84). The essential role of
RA in regulating T-cell gut-tropism was further demonstrated by the fact that ␣4␤7⫹ memory/activated T cells
were significantly reduced in lymphoid organs and depleted
of intestinal LP in VAD mice (84).
Multiple studies have confirmed the direct effect that RA
imposes on ␣4␤7 and CCR9 expression in both CD4⫹ and
CD8⫹ T cells. Using in vitro culture system, Mora et al.
(136) found that CD8⫹ T cells activated by DCs from peripheral LN gain skin-homing tropism by expressing E- and
P-selectin ligands by default, while RA-producing DCs
from the intestine allows for ␣4␤7 and CCR9 expression
(136). Other groups proved that RA induced ␣4␤7 and
CCR9 expression on CD4⫹ T cells while inducing simultaneous FoxP3 expression (11, 139, 181).
1. RA regulation of ␣4␤7
Kang et al. (90) found that RA treatment enhanced transcription of both integrin ␣4 and CCR9 on T cells, whereas
both integrin ␣E and the constitutive expression of ␤7 can
be enhanced by TGF-␤. Both integrin ␣4 and ␣E bind to ␤7.
␣E␤7 binds E-cadherin on epithelial cells (25). Even though
there are seven potential RAREs in ␣4 promoter region,
only one RARE was confirmed by the authors through
binding to RAR␣ in ChIP assay. Therefore, coordination
between TGF-␤ and RA determines T cell migratory ability.
Another study further demonstrated that increasing the relative amount of ␣4 to integrin ␤1 is essential for RA-induced ␣4␤7 expression, as ␣4 predominantly binds to ␤1 in
both naive and activated CD4⫹ T cells and only binds to ␤7
when highly expressed (41). ␣4␤1 is involved in leukocyte
migration to mucosal tissues, bone marrow, splenic follicles, and inflamed tissues (71, 199). The active participation
of ␤1 in RA-regulating ␣4␤7 expression on T cells proved
to be physiologically important as ␤1⫺/⫺ mice showed
lower memory CD4⫹ T cells in bone marrow but elevated
in PP.
2. RA regulation of CCR9
By measuring CCR9 mRNA level in activated CD4⫹ T cells
in the presence of RA, and assessing the binding between
NFAT and RAR-RXR complex in CCR9 promoter regions,
Ohoka et al. (148) demonstrated that transient TCR-mediated signaling and CD28 costimulatory signals induced
transcription factor NFATc1 and NFATc2 nuclear translocation. NFATc2 enhanced but NFATc1 inhibited RARRXR binding to RARE in CCR9 promoter region (148).
Only transient T-cell activation allows for CCR9 upregulation as prolonged TCR signaling retains inhibitory NFATc1
in the nucleus. This finding confirms that the cross-talk
between RAR-RXR and other TCR signaling-induced transcription factors is essential for the delicate regulation of
RA-responsive genes expression. Another elegant study by
Agace group (183) showed that high antigen dose decreased
RA-induced CCR9 expression on activated CD8⫹ T cells,
but induced E-selectin ligand expression. High antigen dose
signal seems to function independently of or downstream of
RA signaling. Thus T-cell receptor (TCR) signaling strength
seems to determine CCR9 expression level. It remains unknown how RA signaling and TCR activation pathway cooperatively determine the tissue-tropism of CD8⫹ T cells.
Of note, CCR9 is constitutively expressed in naive
CD4⫹CD8⫹ and CD8⫹ thymocytes, and on a subset of
CD8⫹ T cells exiting the thymus into lymphoid organs (24).
It remains unknown whether the steady-state CCR9 expression in these cells is regulated by RA signaling. There have
not been studies addressing the mechanism of how CCR9
expression is lost on naive CD8⫹ T cells activated in vitro in
the absence of RA.
The essential role of RA in inducing ␣4␤7 and CCR9 expression was not only demonstrated pharmacologically by
administration of specific RARs agonists/antagonist (including RA), but also by genetic approaches to manipulate
RA signaling mediated in whole mouse or specific cell types.
In line with previously published data using RAR␣ antagonist, both our lab (68, 69, 151) and Hall et al. (70) showed
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that intrinsic RA signaling is required for gut-imprinting of
T cells. Downregultion of RA-induced ␣4␤7 and CCR9
expression was observed in both dnRAR␣-expressing and
RAR␣-deficient CD8⫹ T cells and illustrated the requirement of RAR␣-RXR for CCR9 upregulation (68). In line
with our studies, previous research showed that RAR␥ deficiency did not lead to any loss of RA-induced ␣4␤7 or
CCR9 expression on CD4⫹ or CD8⫹ T cells when activated
in vitro (82).
B. RA-Dependent Induction of FoxP3 Treg
Cells to Mediate Tolerance
It has been proposed that constitutitve RA presence in the
gut is essential for tolerance to commensal microbes and
dietary antigens by inducing Tregs to dampen the potential
proinflammatory responses. To maintain immunity against
foreign pathogens versus tolerance against food antigens,
gut-associated DCs or other APCs have to distinguish, process, and present various antigens to induce inflammatory
T-cell response or Treg differentiation, respectively.
1. Gut-associated Treg differentiation
Numerous studies have demonstrated that RA in combination with TGF-␤ can induce naive CD4⫹ T cells differentiation into stable FoxP3⫹ T cells both in vitro and in vivo
(11, 31, 139, 181). Benson et al. (11) demonstrated that in
vitro RA promoted induced TGF-␤-dependent generation
of inducible Tregs (iTreg) generation even in the presence of
high-level costimulation. In vivo, these Tregs were very stable under CFA-driven inflammation (11). We will refer to
Tregs generated in the presence of RA as RA-Tregs. Another group reported that oral administration of protein
antigen or lymphopenic host induced conversion of naive
CD4⫹ T cells into Tregs in the small intestine. This induction was dependent on RA produced by LP-DCs, because
RAR-specific antagonists LE540 and LE135 inhibited RAinduced FoxP3 expression (181).
Further studies elucidated that different types of APCs are
responsible for inducing RA-dependent Treg differentiation. It was observed that CD103⫹ and not CD103⫺ MLNDCs promoted the conversion of naive T cells into FoxP3⫹
Tregs. This conversion was dependent on both TGF-␤ and
RA (31). Further study showed that indeed bile retinoids
could also imprint intestinal CD103⫹ but not CD103⫺ DCs
with the ability to generate gut tolerance (85). Consistently,
CD103⫹ DCs showed higher transcription of genes related
to the activation and secretion of TGF-␤ (tgfb2, plat, itbp3,
and Raldh2) than CD103⫺ DCs (31). The addition of RA to
CD103⫺ DCs partially allowed these APCs to induce Foxp3
expression in T cells, indicating that RA is not the unique
factor produced by these APCs for inducing Treg conversion.
132
A recent study showed that a deficient production of GMCSF [encoded by colony-stimulating factor (Csf2)] from
granulocyte-macrophages can affect the phagocytic functions of APCs, leading to reduced Treg numbers and impaired oral tolerance (138). Exogenous GM-CSF can restore the immunoregulatory potential of Csf2⫺/⫺ APCs ex
vivo. Notably, blockade of RA abrogated the ability of
exogenous GM-CSF to rescue Treg induction in vitro by
Csf2⫺/⫺ APCs, whereas addition of RA rescued Treg induction in these cultures. Consistent with these findings, injection of a RAR antagonist compromised GM-CSF-mediated
rescue of Tregs in Csf2⫺/⫺ mice in vivo, whereas injection of
RA restored Treg frequency in Csf2⫺/⫺ mice in vivo.
The studies mentioned above describe the importance of
RA in promoting the conversion and stability of Tregs.
However, RA can additionally inhibit the differentiation of
inflammatory Th17 cells. Mucida et al. (139) showed that
TGF-␤ induced Th17 differentiation in the presence of IL-6,
but RA inhibited Th17 differentiation allowing Treg differentiation. Exogenous RA was shown to inhibit induction of
Th17 cells in vivo using an infection model, whereas injection of RAR antagonists resulted in a decrease in
Foxp3⫹Treg cells in the LP (139). In addition, another
study found that intestinal macrophages, by secreting large
amounts of IL-10 and RA, might limit their own production
of IL-6 and IL-23. Thus, in the TGF-␤-rich intestinal microenvironment, LP macrophages would continuously favor the generation of Treg cells (40).
In addition to the prominent role that RA plays in gut
tolerance, it also plays an important role in the immune
privilege of the eye. Retinal pigment epithelial cells were
found to produce both TGF-␤ and RA, thus converting
bystander CD4⫹ T cells into FoxP3⫹ Treg. Both pretreatment with RAR antagonists and silencing of RAR function
in retinal pigment epithelial cells prevented Treg conversion. As anticipated, experimental autoimmune uveitis developed in VAD but not in vitamin A-sufficient mice (94).
2. Role of RA in the induction of FoxP3⫹CD8⫹ Treg
The first observation of RA-induced FoxP3 expression in
CD8⫹ T cells was reported by Kishi et al. using an autoimmune diabetes model (98). The authors generated islet-specific CD8⫹FoxP3⫹CD103⫹ Tregs by stimulating naive
CD8⫹ T cells with splenic DCs in the presence of the specific
peptide, TGF-␤, and RA in vitro. The CD8⫹FoxP3⫹ Tregs
generated in vitro inhibited diabetogenic CD8⫹ T cells proliferation in vitro and completely prevented diabetes onset
when transferred together with diabetogenic splenocytes
from non-obese diabetic (NOD) mice. Using a transgenic
mouse model, which express hemagglutinin (HA) antigen
on intestinal epithelial cells, Fleissner et al. (53) found that
CD8⫹FoxP3⫹ cells can be induced in the MLN. These
CD8⫹FoxP3⫹ cells suppressed both CD4⫹ and CD8⫹ T cell
proliferation (53). Further studies illustrated that RA in
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RETINOIC ACID AND IMMUNITY
combination with TGF-␤ can induce FoxP3 expression on
HA-specific CD8⫹ T cell when stimulated by MLN DCs in
the presence of HA peptide. In the presence of IL-2, TGF-␤,
and RA, donor DCs induced and expanded alloantigenspecific CD8⫹FoxP3⫹ suppressive cells with enhanced expression of CCR4, CTLA4 and CD103. The induced
CD8⫹FoxP3⫹ cells protected skin allografts in vivo via suppressing the upregulation of costimulatory molecules on
DCs, and the production of proinflammatory cytokines by
both CD4⫹ and CD8⫹ T cells (107). Even though
FoxP3⫹CD8⫹ Tregs are not yet well recognized as a major
suppressive population, the above studies illustrate that
RA-dependent induction of FoxP3 expression was similar
in CD4⫹ and CD8⫹ T cells, highlighting its role in maintaining immune tolerance.
3. RA-dependent induction of human Tregs
There have been contradictory findings in the regard of how
RA regulates FoxP3 induction in human cells. Studies using
human peripheral blood cells proved that ATRA and
TGF-␤ converted naive but not memory CD4⫹ T cells into
stable and suppressive FoxP3⫹ Tregs. Furthermore, ATRA
treatment enhanced the suppressive capability of natural
Tregs, reinforcing the key role that RA plays in tolerance
mediated by both natural and induced Tregs (205). Lu et al.
(115) found that RA enhanced and stabilized FoxP3 expression in CD4⫹CD45RA⫹ naive human T cells activated with
suboptimal ␣CD3 and ␣CD28 in the presence of IL-2 and
TGF-␤ in vitro. These induced Tregs showed suppressive
activity in vitro and prevented immunodeficient mice from
human-anti-mouse GVHD (115). Another group revealed
the synergistic effect between ATRA and rapamycin in
maintaining FoxP3 expression and suppression activity of
human Tregs (59). ATRA alone only maintains transient
FoxP3 expression during the expansion of Tregs purified
from adult human peripheral blood. This study suggests the
potential requirement for both rapamycin and ATRA in
generating a large number of stable Tregs for therapeutic
infusion into patients.
4. Molecular mechanism of RA-induced FoxP3
expression
The molecular mechanism of how RA controls FoxP3 expression has been investigated at transcriptional and epigenetic levels. It is well established that the enhancement of
FoxP3 expression by RA depends on TGF-␤, as RA alone is
insufficient to induce Foxp3 expression (11, 31, 139). Studies using neonatal mouse cells and luciferase reporter assasys showed that TGF-␤ triggered the activation of Foxp3
gene, via the binding of Smad3 and NFAT to an enhancer in
the intronic conserved non-coding sequence (CNS1) (195).
Recent reports showed that CNS1 contains binding sites for
transcription factors NFAT, Smad3, and RAR/RXR. Further studies revealed that RA enhances TGF-␤ signaling by
increasing the expression and phosphorylation of Smad3
(146, 217). Indeed, RA actions involve the binding of RAR/
RXR to CNS1. This leads to the increased histone acetylation in the region of the Smad3 binidng site and increased
binding of phosphorylated Smad3, leading to increased
FoxP3 expression (218).
However, other researchers reported that RA could induce
FoxP3 expression even in CD4⫹ T cells isolated from
Smad3 or Smad2 knockout mice. These observations indicate that RA upregulates an alternative signaling axis of
TGF-␤, leading to ERK1/2 rather than Smad2/3 activation
(114). The contradictory findings may be due to redundant
or overlapping signaling pathways in the control of FoxP3
transcription. These findings are also consistent with recent
observations that Smad3 binding to CNS-1 is dispensable
for FoxP3 expression except in the gut (160).
Recent studies by two different groups have shown that
miR10a expression is a signature of RA-induced Tregs. One
study showed that miR10a is highly expressed in natural
Tregs compared with other T-cell subsets and can be induced by TGF-␤ and RA. MiR10a in these RA-induced
Tregs can target both Bcl-6 and Ncor2, and attenuate the
conversion of inducible Tregs into follicular helper T cells
(187). Another study confirmed that miR10a is selectively
upregulated in TGF-␤-induced Tregs only in the presence of
RA, and allows for stable FoxP3 expression; however, genetic ablation of miR10a had no impact on FoxP3 expression. These studies open the question as to how RA regulates various microRNAs, which have been discovered to
regulate various aspects of the immune response. It will be
interesting to elucidate the pathways that RA regulates
through fine-tuning of miRNAs so that it may determine the
stability and plasticity of various T-cell subsets.
C. RA in Regulation of Other Aspects
of T-Cell Differentiation
1. Th17 differentiation
The impact of RA on Th17 survival and differenftaition is
highly RA concentration dependent. Various studies have
shown that RA suppresses Th17 differentiation while promoting TGF-␤-depenent Treg differentiation (139, 217).
Xiao et al. (217) showed that RA inhibited IL-6R, interferon (IFN) regulatory factor 4 (IRF4), and IL-23R, which
are all required for Th17 differnetiation, induced by TGF-␤
during Th17 differentiation, thus suppressing Th17 development (217). Additional studies addressed the molecular
mechanism of how RA regulates Th17 differentiation. In
naive mice, the steady-state ATRA in mouse sera is 1–10
nM (1–10 ␮g in 25 g adult mice) (126, 143). Therefore, we
propose that exogenous RA above 100 nM should be considered a pharmacological concentration. The following
discussion will focus on how the physiological and pharma-
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cological concentration of RA may deliver different conclusions.
A detailed study demonstrated that flagellin-activated LPDCs produce RA to promote both Th17 and Th1 cells differentiation in vitro (196). IL-17 instead of IFN-␥ production is inhibited by RA inhibitor LE540, illustrating the
specific role of endogenous RA produced by LP-DCs in
driving Th17 differentiation. Unsurprisingly, 1 nM RA is
sufficient to promote Th17 differentiation in the presence of
splenic DCs. The reduced IL-17 and IFN-␥ production by
10 ␮M RA (⬃1,000-fold higher than physiological concentration) may indeed be caused by high toxicity. Similarly,
Elias et al. (51) also showed that 1 ␮M Pan-RAR agonist,
4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNBP), inhibited IL-17
differentiation in vitro (51). We suggest that the concentration may have been too high for evaluating the physiological role of RA in Th17 differentiation. Th17 cells are decreased in the SI of VAD mice, whereas DCs isolated from
MLN, PP, or SI-LP of VAD mice, enhanced Th17 cells
differentiation in vitro (26). The discrepany ascribed to the
reduced commensal microbes in the gut of VAD mice, highlighting that Th17 differentiation is fine-tuned by RA in the
gut via various mechanisms. Another study further argued
against the general conclusion that RA suppressed Th17 cell
differentiation, illustrating that although slightly reduced,
Th17 differentiation still occurred in the presence of ⬍30
nM RA, and these Th17 cells generated in vitro proved
proinflammatory in vivo and migrated to the intestine
(203). In the same study, pharmacological levels of RA
(⬎30 nM) inhibited Th17 differentiation in vitro. In other
words, the experimental outcome is largely dependent on
the concentration of RA used in the culture. Therefore, the
controversy between various studies may be due to the concentration of RA and other cytokines applied. To more
accurately assess the role of RA in Th17 differentiation fate,
new insights should be gained through the analysis of Th17
response via genetic blockade of RA signaling. In this regard, Hall et al. (70) and our group reported deficient Th17
cell differentiation in RAR␣-deficient CD4⫹ T cells. In the
study of Hall et al. (70), defective TCR signaling was observed in the RAR␣-deficient CD4⫹ T cells, too. It remains
unknown whether RAR␣ deficiency has any impact on
ROR␥T expression per se.
2. Th1/Th2 differentiation
Earlier studies revealed that VAD mice display excess Th1
and insufficient Th2 response via various mechanisms. One
study followed to demonstrate that the RXR but not RAR
signaling pathway activation enhanced Th2 development
(177). These authors claimed that 9-cis-RA but not ATRA
supplementation enhanced Th2 development from naive
Th0 cells isolated from DO11.10 TCR transgenic mice.
Further studies elucidated that RXR agonist AGN194204
but not RAR agonist TTNPB promoted Th2 development
134
even under ␣CD3 and ␣CD28 stimulation in the absence of
APC. Iwata et al. (83) demonstrated that RA at ⱖ1 nM
directly suppressed Th1 but enhanced Th2 differentiation.
This effect can be mimicked by RAR agonists Am80 and
Tp80, but not RXR agonists HX600 or TZ335. These findings reinforced the role of RAR-dependent RA signaling in
promoting Th2 but suppressing Th1 development (83). In
human T cells, RA, RAR␣ agonist AM580, and not RAR␤/
RAR␥ agonists decrease IFN-␥ and TNF-␣ production
while promoting IL-4, IL-5, and IL-13 synthesis (35).
3. CD8⫹ T-cell differentiation
In an oral immunization model of Listeria monocytogens,
Huang et al. (77) found that RA and TGF-␤ produced by
MLN DCs enhanced the expression of CD8␣␣ on activated
CD8␣␤⫹ T cells. CD8␣␣ can interact with a nonclassical
MHCI molecule thymus leukemia antigen induced on DCs
so that high-affinity memory precursor cells are geared towards the gut-imprinting effector cells (77). This study supports the concept of introducing RA into a vaccination to
induce highly Ag-sensitive CD8⫹ memory cells in the intestine for defense against mucosal pathogens. Our own studies have illustrated that RA in the periphery is required for
CD8⫹ T cell survival during expansion, thus allowing for
the optimal antigen-specific CD8⫹ T-cell expansion and
accumulation (69). Our further study suggested that RAR␣
is the major isoform in determining CD8⫹ T-cell survival
during systemic Listeria monocytogens infection (68).
Taken together with what has been discovered about Th17,
Th1/Th2, and CD8⫹ T -differentiation, the contradictory
observations seem to be due to the usage of various agonists/antagonists in experimental contexts. Caution should
be taken when conclusions are drawn from studies with
⬃10-fold above mouse sera RA. In vitro, the presence or
absence of APC in culture may also influence the final outcome of RA signaling on T-cell differentiation phenotype.
More genetic approaches should be harnessed to understand whether RA signaling-deficient CD4⫹ T cells are less
efficient in differentiation into Th2 cells.
D. RA in Regulation of T-Cell Activation and
Effector Functions
Emerging data suggest that RA is critical for the development of T-cell-mediated immunity. Hall et al. (70) have
discovered that RA is essential for TCR signaling in early
CD4⫹ T-cell activation and controls CD4⫹ T-cell proliferation and differentiation into Th1 and Th17 cells in vitro. In
this study, the authors used T cells from RAR␣ knockout
(KO) mice and showed that RAR␣-deficient CD4⫹ T cells
are defective in calcium influx and ERK phosphorylation
and displayed insufficient activation. The RAR␣ KO mice
showed diminished Th1 and Th17 responses in Toxoplasma gondi infection and failure to control the infection.
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RETINOIC ACID AND IMMUNITY
Our studies using another transgenic model by selectively
overexpressing a dominant negative RAR␣ (dnRAR␣) in T
cells have demonstrated that the RA signaling-deficient
CD4⫹ T cells can proliferate, but fail to differentiate into
Th1 and Th17 cells in vivo (151). The differentiation failure
accounts for the delay in allogenic skin graft rejection. The
discrepancy between the two studies is presently unclear
and is likely due to the means with which RAR signaling
was ablated.
E. RA Regulates T-Cell Survival
In addition to the requirement of RA for the development of
effector T-cell immunity, our own studies have shown that
RA is essential for the survival of effector CD8⫹ T cells
during expansion phase (69). In both tumor and neo-antigen immunization model, our studies showed for the first
time that in vivo, RA signaling is essential for CD8⫹ T cells
survival during expansion at effector phase. Since RA signaling in these studies was selectively disrupted in CD8⫹ T
cells, it proves that it is an intrinsic need of T cells to trigger
RA signaling pathway to survive. As anticipated, RA signaling-sufficient CD8⫹ T cells are essential to control tumor
growth. This was confirmed Listeria monocytogenesis infection model, in which we demonstrated that RAR␣-deficient CD8⫹ T cells failed to accumulate and control bacterial expansion in the spleen (68).
In addition to RAR␣, RAR␥ has also been implicated in
CD8⫹ T-cell biology. It has been shown that overexpression
of human RAR␥, under control of the Lck promoter, elevated the number of CD4⫺/CD8⫹ single positive cells in the
thymus. These data suggest that RAR␥ may be involved in
CD8⫹ T-cell development in the thymus (152). However,
when RAR␥ is deleted from hematopoietic compartment, it
is without effect on CD8 ontogeny (82). Therefore, in our
point of view, RAR␥ does not regulate CD8⫹ T-cell survival.
VI. RA REGULATION OF T-CELL IMMUNITY
IN DISEASES
A. RA in T-Cell-Mediated Anti-tumor
Immunity
1. Extrinsic effect of RA on anti-tumor T-cell immunity
Early studies have shown that a vitamin A-rich diet or RA
treatment can enhance the frequency of tumor-specific cytotoxic effector killer T cells elicited by the vaccination with
irradiated tumor cells (118). Other studies found that in
both tumor-bearing mice (102) and cancer patients (220),
high-dose RA treatment can induce the differentiation of
immature myeloid-derived suppressor cells (MDSC) into
mature DCs, thus diminishing the tolerance of T cells and
generating greater T-cell responses against the tumor. An
earlier study showed that liposomal RA treatment induced
regression of murine acute promyelocytic leukemia in cooperation with adaptive immunity (210). In line with these
findings, combination of a DNA vaccine and RA administration induced robust CD4⫹ and CD8⫹ T-cell immunity
and enabled long-term survival in an acute promyelocytic
leukemia mouse model, although ATRA alone failed to
elicit pronounced T-cell immunity (56). While the studies
above were not specifically designed to understand the role
of RA in mediating enhanced anti-tumor T-cell immunity,
they support the notion that ATRA may be important for
the development of anti-tumor T cell response, thus contributing to the observed efficacy in tumor control.
2. Intrinsic effect of RA on anti-tumor T-cell immunity
Our own studies demonstrated that T cell-intrinsic RA signaling is essential for CD8⫹ T-cell expansion and differentiation to IFN-␥ secretion (69) in response to tumor antigen.
Our studies have shown that as tumors progress, there is a
progressive increase in the tissue concentration of RA at the
tumor site (69). One may question as to whether RA in the
tumor microenvironment (TME) contributes to the tolerant
TME by facilitating the development of Treg or is it essential for the development of T effector functions (11, 31,
139, 181). One may speculate that RA produced by host
DCs in TME can indeed induce Treg conversion, given
heightened suppressive cytokines such as TGF-␤ production by malignant cells (45, 191) or host APCs (28) at late
stage of tumor growth. However, we did not observe dramatic Treg changes in TME when RA signaling was blocked
by dnRAR␣ overexpression in T cells. Additional effort
should be given to the study of RA signaling-deficient Tregs
in TME, to evaluate whether Treg functionality in TME
may be indeed dependent on RA signaling. We summarize
the function of RA in regulating various aspects of antitumor T-cell immunity in FIGURE 2.
B. RA in Anti-infection T-Cell Immunity
VAD in children leads to increased susceptibility to ID and
thus higher morbidity and mortality (173). These epidemiological and clinical findings clearly established that RA is
essential for host resistance to infection. T cells are an essential component in fighting against both primary infections and maintenance of long-term memory response
against infectious agents (214). Presently, vitamin A supplementation is currently used in many developing countries to
reduce childhood mortality (78, 81, 172). The essential role
of both RA and T cells in immune responses against ID
suggests that RA signaling may play a key role in the development of T-cell immunity against infection. However,
while VAD is associated with increased susceptibility to ID,
the effect of vitamin A supplementation on ID control has
been beneficial (200)/neutral (141) or even deleterious
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GUO ET AL.
RA
ImDC
mDC
MDSC
RA
Naïve
CD8+
Naïve
CD4+
TGFβ
?
+IL6?
CTL
aTreg
Tumor cell
Th17
(175). RA may thus impose different impact on anti-infection immunity in response to different infectious agents,
depending on what immune subsets are targeted. For instance, one group showed that RA administration enhanced
LPS-induced nitric oxide synthesis 2 (NOS2) activation in
innate immunity, thus leading to increased mortality in
treated rats (42). In contrast, such an effect was not observed in Listeria monocytogenesis-infected rats (163).
Given the importance of adaptive T-cell immunity in host
protection against infection, our following discussion will
focus on how RA regulates anti-infection T-cell immunity
in different infectious diseases.
1. RA regulation of host resistance and CD8⫹ T-cell
immunity
A) RA CONTROLS HOST RESISTANCE AND MUCOSA-RESIDENT CD8
T-CELL IMMUNITY.
⫹
Pharmacological and genetic approaches
have been used to elucidate the function of RA in control of
CD8⫹ T-cell responses to ID. The gut-homing tropism,
FoxP3 induction, and survival effects that RA exerts on
CD8⫹ T cells can all influence host susceptibility to ID.
Kaufman et al. (92) found that VAD diet diminished the
mucosal CD8⫹ T-cell response elicited by intramuscular
immunization with recombinant adenovirus vaccine vector
expressing OVA (rAd5-OVA). As a consequence, this abolished the protection against oral challenge of Listeria
monocytogenesis overexpressing OVA (92). As expected,
retinyl palmitate or RA administration fully restored the
vaccination-driven gastrointestinal CD8⫹ T-cell response
and protection against challenge. Similarly, in VAD mice
there was a reduced frequency of CD8⫹ T cells in the lower
respiratory tract to a candidate human parainfluenza virus
136
FIGURE 2. The role of RA in regulation of
anti-tumor T-cell immunity. Mature DCs produce high amounts of RA in TME, whereas
RA also enhances the maturation of both immature DCs and suppressive MDSCs. Immature DCs and/or tumor cells may also produce suppressive molecule TGF-␤. RA signaling is required for the expansion/accumulation
of tumor-reactive CD8⫹ T cells, thus promoting tumor surveillance. RA and TGF-␤, however, may promote aTreg accumulation in
TME. Depending on tumor types, the presence of IL-6 in TME may also allow for Th17
differentiation. However, the pro- and antitumor activity of Th17 cells in TME is still a
question of debate.
type 1 vaccine compared with control mice. This study suggested vitamin A (or RA) controls CD8⫹ T-cell response in
mucosa other than the gut (157). Therefore, depending on
specific infectious pathogens, RA signaling may impose
quite different effects on systemic CD8⫹ T-cell response. It
has been proposed that the gut-tropism of T cells imposed
by ATRA has proven beneficial in eliciting more robust
T-cell immunity in the mucosa and better control of both
Salmonella (73) and LCMV infection (189). In both studies,
more effector and memory CD8⫹ T cells were observed in
the gut of ATRA-treated mice.
With the use of genetic models that allow conditional blockade of RA signaling, precise questions can be asked as to the
role of RA on specific leukocyte lineages. In one study using
transgenic mice in which RAR␥ is deleted from the hematopoietic compartment, defective primary and memory
CD8⫹ T-cell response against Listeria monocytogenesis
was observed. It was proposed that this was due to a deficiency in macrophages secreting proinflammatory cytokines such as TNF-␣ and IL-6 (82). Interestingly, a recent
study showed that RA signaling-deficient CD8⫹ T cells are
more prone to the development of memory precurcors instead of short-lived effectors in the context of vaccinia virus
infection (4). Genetic deletion of specific RAR isoforms exclusively in T cells allowed for the dissection of RAR␣,
RAR␤, and RAR␥ in control of anti-infection CD8⫹ T-cell
immunity. Our recent study demonstrated that RAR is essential for survival of CD8⫹ T cells in vivo following Listeria monocytogenesis infection. In contrast, RAR deletion
leads to modest deficiency in Ag-specific CD8⫹ T-cell expansion during infection. The defective survival of RAR␣deficient CD8⫹ T cells leads to a deficiency in control of
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Listeria monocytogenesis expansion in the spleen (68). No
defects were observed in RAR␥-deficient CD8⫹ T cells, further corroborating that lower TNF-␣ and IL-6 production
by the RAR␥-deficient macrophages caused defective CD8⫹
T-cell immunity in a host with RAR␥-deficient hematopoietic comparment.
2. RA regulation of host resistance and CD4⫹ T-cell
immunity
A) TH1/TH2 BALANCE. The balance between Th1 and Th2
responses during CD4⫹ T-cell differentiation is critical for
infection control as it determines the humoral response
against pathogens. Previous studies revealed that VAD environment induced constitutive expression of Th1 cytokines, and limited the humoral response to infection, thus
diminishing the host ability to contain infection through
antibody (Ab) production (23). Investigating Trichinella
spiralis infection in VAD mice, Cantorna et al. (22) found
higher IFN-␥ production and lower frequencies of IL-5producing Th2 cells, which can be restored by the addition
of RA in vitro. These data suggest that RA inhibits Th1 and
promotes Th2 differentiation and survival (22). Even in
animals with normal serum levels of RA, RA was shown to
enhance Th2 but inhibit Th1 responses during tetanus toxoid immunization resulting in higher IL-4:IFN-␥ ratio and
enhanced all anti-tetanus toxoid IgG isotypes. These data
suggest that RA can be used as an adjuvant in generating
more robust humoral response via Th2 skewing in vivo
(116). However, despite the skewed Th2 response discussed
above (22, 83), RA treatment of Ascaris suum-infected
swine promoted profound and enhanced Th1, Th2, and
Treg differentiation in the liver, albeit some may be an
indirect impact from innate immune cells, such as more
recruitment of eosinophils and more inflammatory cytokines produced thereafter (34).
Previous studies using CD4⫹ T cells
from both VAD and RAR␣ KO mice showed increased
frequencies of FoxP3⫹ Tregs, deficient proliferation, and
differentiation into Th1 and Th17 cells in Toxoplasma
gondi model, while no dramatic differences were reported
in Th2 response (70). However, the lower Th1 and Th17
response in vivo led to impaired control of systemic Toxoplasma gondi systemically following oral infection, clearly
demonstrating the essential role of intact RA signaling for
protective CD4⫹ T-cell immunity. As RAR␣ is also deleted
APCs in these mice, such as DCs and myeloid cells, the
defective CD4⫹ T-cell responses in vivo may be partially
due to the intrinsic RA signaling deficiency in T cells. Therefore, additional studies in mice with lineage-specific control
of RA signaling will help to address these issues. It also
remains to be elucidated whether intrinsic RA signaling
controls the balance between suppressive Tregs and inflammatory Th1/Th17 response in various ID circumstances.
The immune system has apparently developed sophisticated
mechanisms to control infection and avoid overt pathology
B) TH1/TH17 RESPONSE.
by balancing the magnitude of the inflammatory response.
No doubt, RA falls into the group of fine-tuning molecules
to keep immune response in balance.
C) TREG REGULATION.
RA also regulates host resistance and
immunity via the enhancement of stable and gut-tropic
Tregs. RA-Tregs are more stable than Tregs generated in the
absence of RA (11). Due to concomitant ␣4␤7 and CCR9
expression, RA-Tregs showed preferential migration to the
gut (11, 181). RA-Tregs are thus also superior in protection
against colitis due to the preferential migration to the gut
(139). The role of RA in generating superior Tregs holds
true during chronic helminth infection. Chronic helminth
infection induced tolerogenic CD11clow DCs which induced
RA signaling-dependent Treg generation, and modulated
the immune response. Depletion of CD11chi DCs favored
the dominance of CD11clow DCs, leading to lower effector
CD4⫹ T-cell proliferation and effector cytokine production
(170). RA treatment also enhanced FoxP3 but inhibited
IL-17 expression in colonic biopsies from patients with ulcerative colitis and colon tissues ex vivo. In addition, RA
also downregulated colon inflammatory responses and
ameliorated trinitrobenzene sulfonic acid-induced murine
colitis (7). RA-Tregs are also superior in resolving CD8⫹ T
cell-mediated acute small intestine inflammation (129).
This study highlighted that the selective migration of RA
imposed on Tregs is key to their local suppressive function.
All the above studies support that RA may ameliorate the
infection-relevant pathology in the gut via generating highly
stable and gut-residential Treg.
C. RA and T-Cell Differentiation in
Transplantation
1. Suppression of effector response
During solid organ transplantation, donor alloantigens are
processed and presented to CD4⫹ T cells, and induce the
latter to activate and produce IL-2 and IFN-␥. The inflammatory cytokines can then induce cytotoxic CD8⫹ effector
T cells, B cells, and macrophages proliferation and differentiation, which can mediate allogeneic tissue and organ
distruction (207). Even though numerous studies have confirmed that CD4⫹ and CD8⫹ effector T cells mediate the
rejection of allografts (169), there is always counterregulation by Tregs (as reviewed in Ref. 202). Therefore, the balance between effector T cells and Tregs determines the kinetics
of rejection or acceptance of allogenic tissues or organs. Direct
regulation of RA on T cell allogenic in GVHD development
has been reported. In a mouse model of acute GVHD, RAR␣specific agonist ER-38925 administration dampened the cytotoxic T lymphocyte (CTL) response, and inhibited Th1-derived serum IL-12 and IFN-␥ and macrophage-derived
TNF-␣. The substantial inhibition of antiallogenic T-cell
responses in ER-38925-treated mice therefore prevented
the development of acute GVHD (75).
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2. RA promotes allogenic effector response
Our recent study revealed a novel function of RA as being
essential for the development of T-cell effector response
against an allogenic skin transplant. In a model where T
cell-intrinsic RA signaling was ablated, deficient Th1/Th17
response led to significantly delayed rejection of allogenic
skin grafts. Our failure to observe an overriding suppression of immunity by RA indicated the dominant role of
endogenous RA in driving effector response than Treg development in this model. These studies provide insightful
understanding of the function of RA in controlling the development of effector T cells in allogenic organ rejection.
3. RA regulates transplantation through Treg
development
Despite the requirement of effector T cell-intrinsic RA signaling for allogenic skin rejection, the administration of RA
agonists induces immune suppression in some circumstances. Prior to the discovery that RA can induce Treg
development, a study in cardiac transplantation demonstrated the immunosuppressive efficacy of RAR␣ agonist in
organ transplantation (164). During acute cardiac transplantation, prophylactic treatment with RAR␣-specific agonist ER-38925 in combination with tacrolimus inhibited
cytotoxic effector cells proliferation and alloantigen-specific inflammatory cytokines IL-2, IL-12 and IFN-␥ production, and thus delayed the rejection of the mouse cardiac
allograft (BALB/c¡C3H/HeN). The inhibition of cytotoxic
response may be due to the induction of Treg development
in this model, although not stated by the authors. Another
study revealed that combination of RA and TGF-␤ indeed
attenuated acute cardiac rejection by promoting Treg and
inhibiting Th17 differentiation (204).
Various studies have now proven that Tregs can be generated ex vivo, efficiently dampen the host immune response
against graft-specific antigens, and thus prevent both acute
and chronic allograft rejection in vivo (87, 216). Numerous
studies have demonstrated the success of RA-Treg in the
prevention of allogenic transplant rejection. Moore et al.
(135) cultured naive T cells with allogenic APCs, TGF-␤,
IL-2, and RA to generate Tregs that were successful in preventing allogenic skin rejection, proving the suppressive
function and potential for therapeutic application of RATreg in allogenic graft rejection (135). In a rat orthotopic
tracheal transplantation model, adoptive transfer of RATregs enhanced TGF-␤ and IL-10 production, whereas
inhibited inflammatory cytokines IL-17, IFN-␥, IL-6, and
MCP-1. RA-Tregs also inhibited the infiltration of effector
T cells into the graft. The balance between anti- and proinflammatory response led to attenuated airway obliteration
and graft fibrication, thus protecting rat trachea allograft
rejection (168). More surprisingly, RA-induced CD8⫹
FoxP3⫹ Tregs were found to induce conventional CD4⫹
FoxP3⫹ Treg conversion and concurrently suppressed
138
CD8⫹ T effector expansion in situ, thus protecting allogenic
skin graft (107). Therefore, RA can induce FoxP3 expression on both CD4⫹ and CD8⫹ T cells, and promote immunosuppression required for allogenic grafts protection.
D. RA and T-Cell Differentiation in
Autoimmunity
As RA regulates various aspects of T-cell function, it is not
surprising that RA also modulates the progression of various autoimmune diseases through the regulation of CD4⫹
or CD8⫹ T cells. Specifically, RA can control the differentiation of CD4⫹ T cells into Th1, Th2, and Th17 which
impact on the intensity and pathological manifestations of
many autoimmune diseases. Furthermore, RA may also regulate the progression of autoimmune diseases via control of
CD8⫹ T-cell differentiation. In the following section, we
focus on how CD4⫹ T-cell differentiation is controlled by
RA signaling towards immunity or tolerance, and thus determine the disease incidence and severity in different autoimmune diseases.
1. RA regulates experimental autoimmune
encephalomyelitis
A) RA SUPPRESSES PATHOGENIC T CELLS IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS.
Early in 1991, long before the discovery of Tregs and Th17 cells, 13-cis-RA was demonstrated to prevent both active and passive experimental autoimmune encephalomyelitis (EAE) development through
the inhibition of T-cell proliferation, IL-2 production, and
effector function (123). The same therapeutic efficacy was
observed in ATRA treatment of myelin basic protein
(MBP)-specific cell-induced EAE. RA was found to increase
IL-4 but decrease IFN-␥, IL-2, and TNF-␣ expression in
addition to inhibition of T-cell proliferation (153). The shift
of Th1 to Th2-like phenotype by RA treatment was also
observed in an MBP-specific human CD4⫹ T cell line, supporting the rationale for retinoids in the treatment of multiple sclerosis (113). Further studies have revealed the role
that RA plays in Th17 cells in the treatment of autoimmune
diseases. One study showed that RA inhibited pathological
Th1 and Th17 cells through downregulation of IRF4,
IL6R␣, and IL23R without altering the frequency of Tregs
development in EAE, thus inhibiting EAE onset and progression (217).
B) RA PROMOTES TREG IN AMELIORATION OF EAE.
The role of RA
in promoting different suppressive Treg subpopulations
makes the rationale that RA modulates the balance between
inflammatory and tolerant response during the development of autoimmunity. A novel CD8␣␣⫹ Treg lineage derived from naive CD4⫹ T cells has been recognized lately as
an important suppressive population generated in situ. Interestingly, RA in combination with TGF-␤ can induce
CD8␣␣⫹ Tregs development from naive CD4⫹ T cells in
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RUNX3-dependent manner, to protect mice against myelin
oligodendrocyte glycoprotein-induced EAE (142). The timing of RA treatment, however, needs to be taken into account as long-term treatment also leads to other cytokine
profile change, which may result in effects opposite from
what may be predicted from the disease onset. Studies
showed that retinoids such as AM80, RAR␣/␤-specific ligand, are effective during the early phase of EAE development. Such an effect was correlated with lower IL-10 production during disease progression. However, continuous
administration failed to inhibit late EAE symptoms (99).
Despite the therapeutic efficacy of retinoids in treatment of
EAE, more studies are warranted to illustrate whether RA
signaling-deficient T cells still facilitate EAE progression. It
is of great importance to understand the role of RA signaling in balance between effector and regulatory T cells during the disease progression for better control of time-dependent RA signaling.
2. RA regulates rheumatoid arthritis
Studies have revealed the essential role of RA in progression
of rheumatoid arthritis progression via balancing between
suppressive Treg and pathogenic Th1/Th17 cell differentiation. An early study in rheumatoid arthritis patients suggested that RA treatment decreased IFN-␥ but not IL-2 or
IL-4 production by CD4⫹ T cells from both healthy and
rheumatoid arthritis patients. These results indicated the
potential application of RA in treatment of rheumatoid arthritis (147). A recent study showed that RA treatment
decreased Th17 and increased Tregs in a murine model of
collagen-induced arthritis (CIA), leading to the inhibition of
both incidence and progression of CIA (103). Further studies revealed that concurrent FoxP3 upregulation and IL-17
downregulation is independent of STAT3 or STAT5 signaling pathway, consistent with previous observations using
conditional deletion of stat5 and stat3 (51). A very recent
study demonstrated that all-trans-retinaldehyde (retinal)
treatment reciprocally suppressed Th17 and promoted Treg
development at both mRNA (decrease of IL-17 and IL-21
but increase of FoxP3, SOCS3, and IL-10) and protein level
in DBA1/J mice with CIA, thus partially contributing to the
attenuation of arthritis (150). It remains to be elucidated
how retinal regulates the balance between Th17 and Tregs
in CIA. All-trans-retinaldehyde demonstrated a function
through phospholipase C/1,4,5-trisphosphate/Ca2⫹ signaling independent of its conversion to RA in some cell types
(29). However, the efficacy observed in the above study was
likely via the conversion to RA. In addition to its direct
impact on Treg induction, RA was found to maintain the
stability of natural Tregs and prevent Th17 differentiation.
This occurs as a result of the downregulation of IL-6R expression and pSTAT3 activation (228). Further in vivo
studies elucidated that adoptive transfer of RA-treated
nTreg suppressed the progression of established CIA in
DBA1 mice. However, nontreated nTreg succumbed to
Th17 differentiation in this chronic inflammatory environment and lost therapeutic efficacy. This proved true even if
nTreg maintained stable FoxP3 expression in vivo and were
originally isolated from CIA mice, indicating the therapeutic potential of RA in rheumatoid arthritis (228).
3. RA regulates uveitis
Eyes are one of the immune privileged sites in human body.
Some studies have revealed the role of RA in maintaining
the immune tolerant microenvironment in the eye through
inhibition of Th1/Th17 response and promotion of Treg
development. Immunization of C57BL/6 mice with human
interphotoreceptor retinoid binding protein peptide 1 to 20
[IRBP(1–20)] elicited experimental autoimmune uveoretinitis
(EAU). Keino et al. (95) reported that intraperitoneal RA
administration dampened the severity of EAU when administrated at 200 ␮g/mouse (⬃20-fold above normal sera RA
concentration, every other day) before the disease onset. No
therapeutic efficacy was observed when RA is administrated at effector phase (95). Oral administration of Am80
at 3 mg/kg (⬃75 ␮g/25 g mouse, ⬃7-fold above naive serum
RA level) every other day, inhibited the severity of EAU,
through downregulation of IL-6R, IL-17A, and IFN-␥, and
reciprocal upregulation of FoxP3 (96). The same effect of
IL-6R upregulation on CD4⫹ T cells and antigen-specific
Th1/Th17 response were observed in RA-treated mice as in
EAE studies despite an unaltered Treg population.
Despite the therapeutic efficacy of RA and synthetic RAR
ligands in EAU treatment, the molecular mechanism of ocular immune privilege was not fully understood until recently. The Caspi group was able to purify naive Foxp3gfp⫺CD4⫹ T cells from FoxP3-GFP mice, and convert them
into highly suppressive Tregs using aqueous humor (AH) in
synergy with TGF-␤ and RA. The Tregs also showed the
upregulation of CD25, GITR, CD103, and CTLA4 (226).
More in-depth analysis revealed that AH stabilized TGF-␤
and RA-induced FoxP3 expression. This study indicated a
dual role of RA in the eye: in vision and immune privilege.
The same group further demonstrated for the first time that
in vivo naive CD4⫹ T cells can be converted to Tregs in a
RA-dependent manner in the eye during specific auto-antigen-induced proliferation. Cornea and retina proved to be
the source of RA for Treg development as both are positive
for all three Raldh isoforms expression and activity (227).
More strikingly, either preimmunization of the host with
IRBP(161–180) or priming of CD4⫹ T cells (CD44hiCD62L⫺)
diminished RA-dependent Treg induction significantly,
thus inducing severe uveitis. Regarding the source of RA,
Kawazoe et al. (94) also demonstrated that retinal pigment
epithelial cells produced RA and, in combination with
TGF-␤, induced bystander T cells conversion into Tregs,
thus maintaining the ocular immune tolerance (94). This
study further reinforced the key role of RA in prevention of
autoimmune uveitis development.
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All the above studies extended the important function of
RA in both vision development and immune privilege (201).
Such findings reinforced the importance of sufficient vitamin A intake in both vision development and the tolerant
ocular microenvironment maintenance.
4. RA regulates T cell-mediated type 1 diabetes
Numerous studies have revealed the functional deficiency
or decrease of Tregs in type I diabetes (T1D) patients (as
reviewed in Ref. 18). Hence, manipulating the immune system to facilitate Treg expansion or shift from pathogenic
effector T cells is predicted to be efficient in T1D treatment.
Van et al. (198) showed the therapeutic efficacy of (500
␮g/mouse, every other day) RA in T1D treatment. In their
study, RA treatment of nonobese diabetic (NOD) mice at
10 wk of age age, when the destructive insulitis already
occurred, increased systemic Treg accumulation, decreased
activated CD8⫹ effector T cell and Th1 cell infiltration into
the islet (198). This combinatorial effect on T cells led to the
regression of diabetes. Of note, the efficacy was completely
dependent on Treg increase as depletion of CD4⫹CD25⫹ T
cells impaired the efficacy. This study sets the foundation
that RA-induced Tregs are essential for suppressing the development of T1D even though the direct inhibition of
ATRA on effector T cells proliferation cannot be excluded.
A subsequent study tested the efficacy of retinoids, etretinate, and ATRA in three different preclinical models of
human T1D (179). In streptozotocin-induced diabetes in
CBA/H mice, both drugs suppressed the diseases progression in terms of decreasing hyperglicemia, concomitant
with lower CD4⫹CD25⫹ effector T cells in the periphery.
ATRA also decreased expression of IFN-␥ and IL-17 at
mRNA level and secretion of IL-6, IFN-␥, and IL-17 upon
restimulation ex vivo. The authors observed the same effect
as in previous studies (198) when RA was administrated in
spontaneous diabetes developed in NOD mice. However,
no efficacy was observed when cyclophosphamide was administered to accelerate diabetogenesis, highlighting the
Treg-dependent ATRA efficacy. The discovery of ATRAinduced CD8⫹ Tregs led to the finding that CD8⫹FoxP3⫹
Tregs generated in vitro were suppressive in vivo and effective in T1D treatment (98).
More importantly, recent studies shed light on the importance of conditioning DCs to promote Treg development
via RA production in T1D treatment. Immunization with
Schistosoma mansoni soluble antigen preparations was
known to protect NOD mice from T1D development (30).
Interestingly, ␻-1, a well-characterized glycoprotein in S.
mansoni antigen, was demonstrated to induce both TGF-␤
and RA production by DCs and induce Treg development in
vitro (221). The mechanism emphasized the intrinsic requirement of RA-induced FoxP3 expression in the therapeutic efficacy of S. mansoni antigens in NOD mice.
140
5. RA regulates other autoimmune diseases
In addition to the well-known autoimmune diseases mentioned above, other studies have elucidated the mechanism
of RA signaling in control or treatment of other autoimmune diseases. Sobel et al. (171) reported that CD4⫹ T cells
in systemic lupus erythematosus patients are defective in
both TGF-␤ and RA signaling, indicating the proinflammatory environment caused by Treg decrease. In another study
on experiment autoimmune myositis, Ohyanagi et al. (149)
found that oral administration of Am80 increased IL-4,
IFN-␥, and IL-10 but not IL-17 production by T cells, and
decreased T-cell infiltration into the muscle, leading to ameliorated inflammation. The multifaceted function of RA in
T-cell differentiation and migration makes it a good candidate for the treatment of a wide spectrum of autoimmune
diseases.
Despite the rapid progress in understanding both the mechanism and therapeutic application of RA in control of autoimmune diseases, additional insights are critical. At this
time, we do not have a clear mechanistic understanding of
the molecular impact of RA on key cellular elements (T, B,
myeloid) in vivo and how it governs the development of
tolerance and autoimmunity.
E. RA in T-Cell Regulation for Allergy
Response
The promiscuous impacts of RA on T-cell differentiation
make it an attractive mediator in control of allergy. Early
studies established that T cells in VAD hosts were skewed
towards IL-10⫹ Tregs but diverted from IFN-␥⫹ Th1 memory cells without any impact on IL-4 production (176). In
the following section, we will discuss the role of RA signaling in the control of allergic response via regulation of different T-cell subsets.
1. RA regulates allergy response via Th1/Th2
Various studies have revealed that RA may affect allergy
response by altering both Th1 and Th2 response. Studies
have shown that both ATRA and 9-cis-RA induced Th2
cytokines IL-4 and IL-5 production by human CD4⫹ T
cells, while inhibiting Th1 cytokine IFN-␥ production (36).
As discussed above, the timing of ATRA administration
determines the impact on allergy progression, as CD4⫹ Tcell differentiation status is different at the onset and progression stage. For instance, liposomally encapsulated
ATRA increased IgE response, bronchoalveolar lavage
(BAL) eosinophilia, and accumulation of IL-5, TGF-␤1,
and CCL1 when given at the time of systemic sensitizing
injections for asthma manifestation. However, such variables were not affected when administered at the time of
intranasal challenges (121). In both cases, T cells were
prone to IL-4/IL-5⫹ Th2 but not IFN-␥⫹ Th1 cells in vitro,
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exacerbating allergic and inflammatory response. The different observations at different time points suggest that the
impact on T-cell differentiation, for instance, is dependent
on the priming status (plasticity) of these cells and their
migration into the lung. This was consistent with previous
reports showing that in ATRA-treated acute promyelocytic
leukemia patients, there was a hyperleukocytic response
associated with asthmalike respiratory syndrome (66, 74),
eosinophilia, basophilia and hyperhistaminemia, peripheral
edema, and so on (74).
2. RA regulates allergy through Tregs
More recent studies have focused on the role of RA-Treg in
regulation of T cell-mediated allergy. The ability of ATRA
to induce FoxP3 expression was not only demonstrated in
naive CD4⫹ T cells (11, 139, 181), but also proven true in
Th2 memory cells generated in vivo. Kim et al. (97) discovered that OVA-specific Th2 memory cells generated in vivo
can be converted into functional FoxP3⫹ Tregs in the presence of TGF-␤ and ATRA ⫾ rapamycin under both Agspecific stimulation and polyclonal stimulation (97). The
“converted” Tregs downregulated GATA3 and IRF4; produced little IL-4, IL-5, and IL-13; and migrated efficiently
into the lung. Thus Tregs efficiently suppressed Th2-mediated airway hyperreactivity, including eosinophil and IgE
response. This finding supported the therapeutic potential
of RA in allergy treatment.
In addition to pharmacological approaches, genetic-engineered mouse models were also used to elucidate molecular
mechanisms of how endogenous RA signaling regulates allergy response. In complete Aspergillus allergen-immunized
mice model, epithelial metalloproteinase 7 (MMP7) activated IL-25 to promote Th2 cells differentiation, but inhibited RALDH1 and thus Treg development (61). Therefore,
MMP7⫺/⫺ mice showed higher RALDH1 expression, lower
IL-4 and IL-5 in bronchoalveolar fluid, and dampened airway hyperreactivity. As such, the RALDH inhibitor Citral
restored asthma in MMP7⫺/⫺ mice, whereas ATRA administration attenuated the asthma phenotype with a concomitant increase in Treg.
The key role of RA in modulating allergy via Treg induction
was also demonstrated in Th17-dominated chronic allergic
airway inflammation. During mucosal sensitization with
OVA and TNF-␣, prolonged ATRA treatment enhanced
Treg activity in the lung and consequently ameliorated
Th17-mediated disease, reinforcing the therapeutic potential of ATRA in asthma treatment by modulating Th17Treg balance (225). Of note, ATRA concentration needs to
be taken into account for allergy treatment. In both OVA
immunization-OVA challenge and house dust mite immunization-house dust mite challenge models, different doses
of ATRA changed the allergy response differentially (124).
Even though administration of ATRA at different doses
(50, 500, and 2,500 ␮g) all enhanced eosinophil recruitment to the BAL, fewer eosinophils were recruited into BAL
of 2,500 ␮g ATRA-treated mice than those treated at lower
doses. In addition, total IL-5 and IgE in BAL were decreased
in 2,500 ␮g ATRA-treated but increased in lower dosetreated hosts. Given that 50 ␮g is about fivefold higher
than the total RA concentration in the whole mouse
body, 500 and 2,500 ␮g (⬃50- and ⬃500-fold higher
than physiological concentration, respectively) may indeed have some off-target effects and/or even toxicity.
The lower eosinophil counts in 2,500 ␮g treatent group
may be due to high toxicity. Therefore, lower pharmacological doses of ATRA enhance Th2 response in vivo. The
global adverse effect of high-dose treatment needs to be
taken into account when evaluating the application of
RA in allergy treatment.
More studies are certainly needed to understand the regulatory role of RA in Th2 or Th17-dominant allergy disease.
The recruitment, proliferation, differentiation, and activity
of various T-cell subsets are the key components that RA
signaling may impose impact on during both disease development and treatment.
F. RA and T Cell-Dependent Humoral
Response
As discussed above, VAD leads to increased susceptibility to
ID among young children, causing higher mortality and
morbidity (173). B cells and humoral response are vital to
host protection and vaccination against many infectious
pathogens. There are two types of Ab response, namely,
T-dependent and -independent response, depending on the
specific Ag. Due to the wide-sepctrum features that RA regulates in T-cell differentiation, RA may also modulate humoral response via the pathway of T-cell differentiation. For
researchers interested in the direct impact of RA on B cells,
B-cell differentiation, memory B-cell generation, and IgA class
switching, we recommend other review articles (155).
VII. CONCLUDING REMARKS
Numerous studies have elucidated the mechanism of how
vitamin A controls the immune response through RA
signaling. During embryogenesis, maternally derived RA
is essential for the development of secondary lymphoid
organs (197). In adult mammals, RA controls various
immune cells differentiation and thus the ultimate outcome of immune response and host protection (84, 137).
The dual role of RA promoting tolerance (11, 31, 139)
and immunity (69, 70, 151) in different disease contexts
points to the key notion that RA functions as a regulator
of immune response. The overall impact of vitamin A
deprivation or RA signaling ablation depends on the microenvironment in which other cytokines/chemokines
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may interact with RA-mediated signaling to determine
the cell differentiation fate.
Although Iwata et al. (84) provided the initial clues that the
“anti-infective” effect of vitamin A is conveyed through the
upregulating impact of the gut-homing molecules ␣4␤7 and
CCR9, the impact of RA on immunity is far more profound.
In the periphery, RA controls the specification and fate of
Th1s, Th17s, and Th2s. In this regard, many questions
remain to be answered. First, does RA signaling control the
plasticity and stability of effector T cells during differentiation as it stablizes FoxP3 expression in Tregs? That is, is RA
signaling required for stable expression of GATA-3, T-bet,
and ROR␥T expression possibly through epigenetic modification? Second, what is the difference between signaling
pathways induced by physiological and pharmacological
RA? Caution should be taken in the interpretation of data
acquired through the administration of high-dose ATRA or
9-cis-RA due to possible toxicities. Third, what other transcription factors cooperate with RARs in determining the
lineage commitment of different immune cells? Fourth,
what genes in T cells are directly or indirectly regulated
by RA that impact on their function and fate? To answer
these two questions, advanced proteomic and genomic
analyses need to be harnessed. Chromatin-immunoprecipitation (ChIP)-Seq analysis will help to identify novel
target genes during T-cell differentiation. These mechanistic studies will lead to a better understanding of how
RA exerts such profound control over the polarity of the
immune response.
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence: R. J.
Noelle, Norris Cotton Cancer Center, One Medical Center
Dr., Rubin Bldg., Rm. 730, Lebanon, NH 03755 (e-mail:
RJN@Dartmouth.edu) or MRC Centre for Transplantation, King’s College London, 5th Floor Tower Wing, Guy’s
Hospital, London SE1 9RT, UK (e-mail: Randolph.Noelle
@kcl.ac.uk).
GRANTS
This work was supported by National Institutes of Health
Grant 1R01AT005382.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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