Nutrition xxx (2015) 16

Contents lists available at ScienceDirect

Nutrition
journal homepage: www.nutritionjrnl.com

Basic nutritional investigation

Vitamin A supplementation leads to increases in regulatory

CD4Foxp3LAP T cells in mice
Samara R. Medeiros Ph.D. a, Natalia Pinheiro-Rosa B.Sc. a, Luisa Lemos M.A. a,
Flavia G. Loli B.Sc. a, Alline G. Pereira B.Sc. a, Andrezza F. Santiago Ph.D. a,
Ester C. Pinter a, Andrea C. Alves Ph.D. a, Jamil S. Oliveira B.Sc. a,
Denise C. Cara Ph.D. b, Tatiani U. Maioli Ph.D. c, Ana Maria C. Faria M.D., Ph.D. a, *

gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Departamento de Bioqumica e Imunologia, Instituto de Ciencias Biolo
gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
Departamento de Morfologia, Instituto de Ciencias Biolo
c
~o, Escola de Enfermagem, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
Departamento de Nutrica
a

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 14 January 2015
Accepted 18 March 2015

Dietary compounds, including micronutrients such as vitamin A and its metabolite retinoic acid,
directly inuence the development and function of the immune system. In this study, we show
that either dietary deciency of or supplementation with vitamin A had immunologic effects in
mice that were fed these diets during their development (for 8 wk during the postweaning period).
Decient mice presented higher levels of interferon-g, interleukin (IL)-6, transforming growth
factor-b, IL-17, and IL-10 in the gut-associated lymphoid tissues and draining lymph nodes, indicating a proinammatory shift in the gut mucosa. Serum immunoglobulin G levels also were
elevated in these mice. Conversely, supplemented mice showed higher frequencies of
CD4Foxp3LAP regulatory T cells in gut lymphoid tissues and spleen, suggesting that vitamin A
supplementation in the diet may be benecial in pathologic situations such as inammatory bowel
diseases.
2015 Elsevier Inc. All rights reserved.

Keywords:
Vitamin A
Retinoic acid
Regulatory T cells
TGF-b

Introduction
Dietary compounds directly inuence the development and
the function of the immune system. Micronutrients, such as vitamins, have been described as important factors that affect both
innate and adaptive immune responses. This is the case of
vitamin A, which is consumed in the form of retinol or b-carotene and metabolized to retinoic acid (RA), the active form of the
nutrient [1].
Despite being found in many kinds of food, vitamin A deciency is a global health problem [2]. Vitamin A deciency may
result in higher infant mortality, poor body development, and an
increased susceptibility to infections. These alterations are

mostly due to its key role in epithelial cell differentiation and

integrity. The immune system is also affected by vitamin A
because RA is involved in switching immune response prole
from T helper (Th)1 to Th2, reducing Th17 differentiation in vitro,
in the differentiation of mucosal CD4CD25Foxp3 Tregs, and
in immunoglobulin (Ig)A production in the gut mucosa [35]. RA
also imprints gut-homing properties to activated T and B cells [6].
The aim of this study was to analyze the clinical, biochemical,
and immunologic effects brought about by either dietary deciency of or dietary supplementation with vitamin A in mice
from weaning to adulthood.

Materials and methods

Animals, experimental design, and diets
Female C57 BL/6 mice were obtained from Centro de Bioterismo do Instituto
gicas (CeBioICB), Universidade Federal de Minas Gerais at day
de Ciencias Biolo
21 after birth. Animals were randomly divided in three groups that were fed diets
containing different amounts of vitamin A: no vitamin A (VitA-decient group;
n 5), 4000 IU (control group; n 6), 10 000 IU (VitA-supplemented group;

This study was nancially supported by scholarships and research fellowships

 gico (CNPq)
from Conselho Nacional de Desenvolvimento Cientco e Tecnolo
~o de Aperfeicoamento de
(SRM, AMF, DCC, NPR, FLG, and ACA), Coordenaca

Pessoal de Nvel Superior (CAPES) (LL and AGP), and Fundac~
ao de Amparo a
Pesquisa do Estado de Minas Gerais (FAPEMIG) (AFS), Brazil.
* Corresponding author. Tel.: 55 313 499 2640; fax: 55 313 409 2613.
E-mail address: afaria@icb.ufmg.br (A. M. C. Faria).
http://dx.doi.org/10.1016/j.nut.2015.03.004
0899-9007/ 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Medeiros SR, et al., Vitamin A supplementation leads to increases in regulatory CD4Foxp3LAP T cells
in mice, Nutrition (2015), http://dx.doi.org/10.1016/j.nut.2015.03.004

S. R. Medeiros et al. / Nutrition xxx (2015) 16

20

800

15

600

*
10

400

200

Retinol (ng/100 mg)

Retinol (ng/100 mL)

0
Serum

Control

VitA deficient

Delta animals weight (g)

10
8
6
4
2
0
1

Liver

VitA supplemented

Control
VitA deficient
VitA supplemented

-2

Wk

Fig. 1. Effect of vitamin A deciency or supplementation in weight gain. After weaning (day 21 after birth), mice were fed either vitamin A-decient or vitamin A-supplemented diets for 8 wk. Control mice received standard AIN93 G diet. The mice were weighed weekly. (A) Vitamin A levels were measured by high-performance liquid
chromatography in liver and serum. Bars represent the mean  SEM of a group (n 6). (B) Weekly weight measurement of mice were performed and dots represent the
mean SD of a group (n 6). Differences among groups were calculated using students t test, and statistical signicance is represented by * (P < 0.05), y(P < 0.001), and
z
(P < 0.0001).

n 7). All mice were fed AIN93 G diet (modied or not) for 8 wk from weaning to
adulthood (12 wk of age) following a previously established protocol [7].
Intestinal tissue preparation and cytokine assay
Animals were euthanized by cervical dislocation under anesthesia. After intestine harvesting, colonic tissue was placed in buffer solution with protease
inhibitor (1 mL/0.1 g). Tissue fragments were homogenized and centrifuged and
supernatants were collected for cytokine assay. Interleukin (IL)-10, IL-6, transforming growth factor (TGF)-b, interferon (IFN)-g and IL-17 were measured by
enzyme-linked immunosorbent assay (ELISA) as previously described [8]. All
reagents were obtained from BD-Pharmingen (Franklin Lakes, NJ, USA).
Histopathologic analysis
Colon was xed in 10% formalin in 0.01 M phosphate buffer (pH 7.2) and
embedded in parafn. Sections were stained with hematoxylin and eosin for
histopathologic examination by light microscopy as described previously [9]. For
histologic analysis, the mucosal architecture, muscle thickening, goblet cell
depletion, crypt abscesses, and inltrating cell counts were evaluated and photographed using a digital camera (Moticam 2500, China) coupled with an optical
microscope (Olympus Optical Co., Japan).
Collection of sera, intestinal lavage uid, and immunoglobulin measurement
Blood samples were collected from the axillary plexus in mice under anesthesia, and sera separated. Intestinal lavage uids were collected after euthanasia
ushing the entire intestine with cold-buffered saline (phosphate-buffered saline). Serum Igs (IgG, IgM, and IgA) as well as secretory IgA in the intestinal uid
were measured by sandwich ELISA as previously described [8] using antibodies
from Southern Biotechnology (Birmingham, AL, USA).
Cell preparation and ow cytometry analysis
Cells of lamina propria were isolated by a modied version of a previously
reported method [10], as described elsewhere [8]. Spleen and mesenteric lymph
nodes (MLN) cells were also isolated. Cells from lamina propria compartments,
MLN, and spleen were surface labeled with Fluorescein isothiocyanateconjugated antimouse CD4, a combination of biotin antimouse latencyassociated peptide (LAP) and allophycocyanin-conjugated streptavidin and
intracellularly with phycoerythrin-conjugated antimouse Foxp3 (BD Pharmingen). Labeled cells were acquired using FACScalibur (Becton Dickinson, Mountain
View, CA, USA). At least 30 000 events were counted and data were analyzed
using Flow Jo software.
High-pressure liquid chromatography
For high-pressure liquid chromatography assay, a C18 column was used with
acetonitrile/dichloromethane/methanol (70/20/10) as mobile phase. Plasma and
liver samples were treated with ethanol and n-hexane and dried under nitrogen
as previously described [11].

Statistical analyses
Differences among groups were determined by analysis of variance test,
followed by Tukey test. P < 0.05 was considered significant. Software GraphPad
Prism, version 6 was used for statistical analysis and graph plotting.

Results
Vitamin A deciency does not induce changes in body weight
To evaluate the amount of vitamin A absorbed under either
deciency or supplementation dietary conditions, the concentration of vitamin A was measured in both the serum and in the
liver. As expected, the storage levels of vitamin A in the liver of
decient mice were very low, and levels in the supplemented
animals were higher compared with control mice. Serum levels
of vitamin A in animals from control and supplemented groups
were similar, although a reduction was observed in the decient
group (Fig. 1A).
To study clinical changes that vitamin A deciency and supplementation could bring about, animals weight was veried
weekly during the 8-wk experimental period. There was no
signicant difference in the weight gain among the animals
(Fig. 1B). There was no difference in food intake during the whole
experimental period either (data not shown). Because vitamin A
levels were altered in the livers of supplemented and decient
mice, we evaluated basic biochemical parameters to examine
whether those alterations lead to disturbances in serum
cholesterol, triacylglycerols, and glucose levels as well as in liver
lipid concentrations, but no difference was observed in any of the
parameters evaluated (data not shown).
Vitamin A deciency did not affect mucosal architecture
It is well known that vitamin A is important to maintain the
integrity of intestinal epithelia [12]. We performed colon histologic analysis to study whether 8-wk vitamin A deciency would
lead to morphologic changes in the colon mucosa. No alteration
was found in mice that were fed vitamin A-decient or vitamin
A-supplemented diets (Fig. 2). The thickness of muscle layer or
mucosa muscular was similar in all groups. The submucosal was
thin with no signs of either edema or inammatory inltrated
cells. All groups showed mucosa with globet cells, intestinal cells,
and absence of crypt abscesses.

Please cite this article in press as: Medeiros SR, et al., Vitamin A supplementation leads to increases in regulatory CD4Foxp3LAP T cells
in mice, Nutrition (2015), http://dx.doi.org/10.1016/j.nut.2015.03.004

S. R. Medeiros et al. / Nutrition xxx (2015) 16

Fig. 2. Morphologic study of the colonic mucosa of mice fed either vitamin A-decient or vitamin A-supplemented diets. Colonic tissues were collected, xed in buffered
saline, cut in transversal sections, and stained with hematoxylin and eosin. To evaluate the effects of vitamin A in the colon, the following parameters were analyzed:
maintenance of mucosal architecture, depletion of goblet cells, cellular inltrate in the submucosal layer, thickening of muscle layer, and presence of crypt abscesses. (A, B)
control group; (C, D) vitamin A-decient group, and (E, F) vitamin A-supplemented group. Short arrows indicate the muscle layer and long arrow indicate goblet cells (40).
The bars indicate (A, C, E) 100 mm or (B, D, F) 20 mm.

Vitamin A deciency led to elevated serum IgG levels and

increased cytokine production at mucosal sites

Vitamin A supplementation led to increased frequencies of

CD4Foxp3LAP regulatory T cells

To evaluate the effect of vitamin A in immune cell function,

serum and secretory Ig levels as well as levels of cytokines in gutassociated lymphoid tissues were measured. Serum IgG levels
were elevated in vitamin A-decient mice but unaltered in supplemented mice compared with control animals (Fig. 3A). Secretory IgA (Fig. 3A) as well as serum IgM and IgA levels were not
affect by vitamin A deciency or supplementation (data not
shown). Production of IFN-g, IL-17, IL-6, TGF-b, and IL-10 (Fig. 3B
F) was increased in the colonic tissues of decient mice, IFN-g
(Fig. 3B) and IL-6 (Fig. 3D) levels were also increased in MLN of
vitamin A-decient mice. A reduction was observed in the production of IL-17 in the MLN of the supplemented group (Fig. 3C).
No alterations in cytokine production were detected in the spleen.

Because vitamin A is involved in the differentiation of Foxp3

regulatory T (Tregs) cells in the gut mucosa, we next analyzed the
frequency of Tregs expressing Foxp3 and the surface form of TGFb (LAP cells). There was an increase in the frequency of
CD4Foxp3LAP cells in spleen, MLN, Peyers patches, and
colon lamina propria of vitamin A-supplemented mice compared
with both control and decient mice (Fig. 4).
Discussion
In this study, we examined the clinical, biochemical, and
immunologic effects of either dietary deciency of or supplementation with vitamin A in mice during their development (for

Please cite this article in press as: Medeiros SR, et al., Vitamin A supplementation leads to increases in regulatory CD4Foxp3LAP T cells
in mice, Nutrition (2015), http://dx.doi.org/10.1016/j.nut.2015.03.004

S. R. Medeiros et al. / Nutrition xxx (2015) 16

B
5

3
2

50 000

1
0

0.4

Colon

4
2

Spleen

Colon

MLN

1
0

Spleen

MLN

0
Spleen

MLN

0.2
0.0

0
Colon

TGF- (ng/mL)

IL-6 (ng/mL)

0.1

Spleen

2.0
1.5
1.0
0.6

0.0

IL-10 (ng/mL)

IgG (g/mL)

100 000

Secretory IgA (g/mL)

0.8
0.6
0.4
0.2
0.2

IL-17 (ng/mL)

IFN- (ng/mL)

150 000

Control

Colon

VitA deficient

MLN

Spleen

Colon

MLN

VitA supplemented

Fig. 3. Effect of vitamin A deciency and supplementation on Ig secretion and cytokine production in gut-associated lymphoid tissues. Serum Igs and secretory IgA in the
intestinal uid (A) was measured by sandwich ELISA. MLN, colonic tissues, and spleen were collected and homogenized in buffered saline with protease inhibitors. Cytokines
in the supernatants of tissue homogenates (BF) were measured by ELISA. Bars represent mean SEM of a group (n 7). Differences among groups were calculated using
ANOVA, and statistical signicance is represented by *(P < 0.05), y(P < 0.01), and z(P < 0.001). ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; IFN,
interferon; Ig, immunoglobulin; IL, interleukin; MLN, mesenteric lymph node.

8 wk in the postweaning period). Initially, we examined the effects of dietary manipulation in serum and liver storage levels of
the hydrophobic vitamin. All mice, including those that were fed
a vitamin A-decient diet after weaning, received a supply of
vitamin A by breast-feeding because mothers milk is one of the
major sources of vitamin A. This is probably why they had a
signicant stock of this vitamin in the liver at the beginning of
the experiment. However, 8 wk of insufcient ingestion of
vitamin A was enough to lower the level of this nutrient in liver
and serum. Despite that, none of the dietary manipulations
induced changes in weight gain. Vitamin A is important for the
epithelial growth and maintenance [12], but the period of
deprivation was not enough to induce histologically detectable
disturbances in the gut mucosa (Fig. 2). The only change noticed
was a slight augmentation in the size of epithelial-related goblet
cells in supplemented mice (Fig. 2C).
Although dietary modications in vitamin A content did not
lead to changes in the gut mucosa architecture, some immune
functions were altered mostly in the gut-associated lymphoid
tissue and draining lymph nodes. All cytokines measured (IFN-g,
IL-6, IL-17, TGF-b, and IL-10) were increased in the colon of
vitamin A-decient mice. IFN-g and IL-6 were also increased in
the MLN of these mice. Cytokine IL-6, together with TGF-b, has
an important role in driving the differentiation of Th17 cells [6,
13]. As both cytokines were increased in the colon, it is plausible that the higher levels of IL-17 were due to an enhanced
generation of Th17 cells. Levels of the anti-inammatory cytokine IL-10 were also increased in the colon, suggesting a
compensatory loop provided by the local inammatory conditions as previously shown in experimental models of colitis [14]

and asthma [15]. Along with this proinammatory prole of

cytokine production, serum IgG levels were elevated in decient
mice. IgG production is dependent on IFN-g secretion [16] by T
cells and, although there is no change in the levels of this cytokine in spleens of decient mice, it is possible that the higher
levels of IFN-g in mucosal associated organs had an effect in the
systemic levels of this immunoglobulin. Despite the alteration in
the cytokine prole in the gut mucosal tissues, it did not inuence secretory IgA production. Levels of TGF-b were higher in
decient mice and this cytokine is critical for IgA production [17],
therefore the increase in inammatory cytokines may have not
been enough to interfere with secretory IgA secretion in the gut.
On the other hand, supplementation with vitamin A led to an
increase in the frequency of a subpopulation of Treg cell
expressing a membrane bound form of TGF-b associated with
LAP [18]. It was already shown that RA, a metabolite of vitamin A,
can be produced by CD103 dendritic cells expressing the
enzyme RALDH in the intestinal mucosa. RA acts along with TGFb in the differentiation of nave CD4T cells into regulatory
CD4Foxp3T cells in vitro [5] and in vivo [19]. To our knowledge, this study demonstrated for the rst time that supplementation of this nutrient in the diet for 8 wk could increase the
population of regulatory CD4Foxp3LAPT lymphocytes in all
organs analyzed, but no difference was observed in the whole
population of CD4Foxp3T cells. It is possible that in fully
developed mice at physiological conditions, CD4Foxp3Tregs
are already at their optimal frequencies. However,
CD4Foxp3LAPT cells could represent a subset among
Foxp3Tregs that are more sensitive to the effects of vitamin A,
and therefore were the one affected by its increased levels.

Please cite this article in press as: Medeiros SR, et al., Vitamin A supplementation leads to increases in regulatory CD4Foxp3LAP T cells
in mice, Nutrition (2015), http://dx.doi.org/10.1016/j.nut.2015.03.004

S. R. Medeiros et al. / Nutrition xxx (2015) 16

Spleen

% CD4+FoxP3+LAP+

15

C
40

% CD4+FoxP3+LAP+

MLN

__________________

_________

10

30
20
10

Peyer's patches

% CD4+FoxP3+LAP+

60

Colon lamina propria

__________________
_________
*

60

% CD4+FoxP3+LAP+

__________________
*
_________
*

40

20

__________________

_________

40

20

Control

VitA deficient

VitA supplemented

Fig. 4. Effect of vitamin A on the frequencies of regulatory CD4Foxp3LAP T cells. Cells from colonic lamina propria, MLN, Peyers patches, and spleen were isolated,
stained with uorochrome-labeled antibodies and analyzed by ow cytometry. Frequencies of CD4Foxp3LAP cells in spleen (B), MLN (C), Peyers patches (D), and colon
lamina propria (E) were analyzed using the lymphocyte gate (A). Triangles, squares, and circles represent individual animals and lines represent the mean of a group (n 4
7). Differences among groups were calculated using ANOVA test. Signicant differences are represented by * (P < 0.05), y(P < 0.01), and z(P < 0.0001). ANOVA, analysis of
variance; LAP, latency-associated peptide; MLN, mesenteric lymph node.

Conclusion

Acknowledgment

Vitamin A is essential to the maintenance of the homeostasis of

gut lymphoid cells. In this study, its deciency led to disturbances
in inammatory cytokines in the gut mucosa as well as in systemic
Ig production. Conversely, to our knowledge, we showed for the
rst time, that vitamin A supplementation of even healthy mice
boosted the differentiation of CD4Foxp3LAPTregs in local
and systemic lymphoid organs. Therefore, this dietary manipulation may help to boost Treg responses in pathologic conditions
such as inammatory bowel diseases.

The authors acknowledge Ilda Marcal de Sousa for the

excellent care of the mice.
References
[1] Faria AM, Gomes-Santos AC, Goncalves JL, Moreira TG, Medeiros SR,
Dourado LP, et al. Food components and the immune system: from tonic
agents to allergens. Front Immunol 2013;4:102.
[2] FAO/WHO. Human vitamin and mineral requirements. In: Human Vitamin
and Mineral Requirements. [s.l:s.n.]. 2009:87107. Available at: http://
www.fao.org/3/a-y2809e.pdf.

Please cite this article in press as: Medeiros SR, et al., Vitamin A supplementation leads to increases in regulatory CD4Foxp3LAP T cells
in mice, Nutrition (2015), http://dx.doi.org/10.1016/j.nut.2015.03.004

S. R. Medeiros et al. / Nutrition xxx (2015) 16

[3] Mora JR, Iwata M, Von Andrian UH. Vitamin effects on the immune system:
vitamins A and D take centre stage. Nat Rev Immunol 2008;8:68598.
[4] Mucida D, Park Y, Kim G, Turovskaya O, Scott I, Kronenberg M, et al.
Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic
acid. Science 2007;317:25660.
[5] Mucida D, Cheroutre H. TGFb and retinoic acid intersect in immuneregulation. Cell Adh Migr 2007;1:1424.
[6] Benson MJ, Pino-Lagos K, Rosemblatt M, Noelle RJ. All-trans retinoic
acid mediates enhanced T reg cell growth, differentiation, and gut
homing in the face of high levels of co-stimulation. J Exp Med
2007;204:176574.
[7] Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 puried diets for laboratory
rodents: nal report of the American Institute of Nutrition ad hoc writing
committee on the reformulation of the AIN-76 A rodent diet. J Nutr
1993;123:193951.
[8] Gomes-Santos AC, Moreira TG, Castro-Junior AB, Horta BC, Lemos L,
Cruz DN, et al. New insights into the immunological changes in IL-10decient mice during the course of spontaneous inammation in the gut
mucosa. Clin Dev Immunol 2012;2012:560817.
[9] Lev R, Spicer SS. Specic staining of sulphate groups with alcian blue at low
pH. J Histochem Cytochem 1964;12:309.
[10] Davies MD, Parrott DM. Preparation and purication of lymphocytes from
pria of murine small intestine. Gut
the epithelium and lamina pro
1981;22:4818.
[11] Arnaud J, Fortis I, Blachier S, Kia D, Favier A. Simultaneous determination
of retinol, alpha-tocopherol and beta-carotene in serum by isocratic

[12]

[13]

[14]
[15]

[16]
[17]

[18]

[19]

high-performance liquid chromatography. J Chromatogr 1991;572:

10316.
Osanai M, Nishikiori N, Murata M, Chiba H, Kojima T, Sawada N. Cellular
retinoic acid bioavailability determines epithelial integrity: role of retinoic
acid receptor alpha-agonists in colitis. Mol Pharmacol 2007;71:2508.
Fina D, Sarra M, Fantini MC, Rizzo A, Caruso R, Caprioli F, et al. Regulation of
gut inammation and th17 cell response by interleukin-21. Gastroenterology 2008;134:103848.
Strober W, Fuss I, Mannon P. The fundamental basis of inammatory bowel
disease. J Clin Invest 2007;117:51421.
Faustino L, Mucida D, Keller AC, Demengeot J, Bortoluci K, Sardinha LR,
et al. Regulatory T cells accumulate in the lung allergic inammation and
efciently suppress T-cell proliferation but not Th2 cytokine production.
Clin Dev Immunol 2012;2012:721817.
Stavnezer J, Guikema JE, Schrader CE. Mechanism and regulation of class
switch recombination. Annu Rev Immunol 2008;26:26192.
Coffman RL, Lebman DA, Shrader B. Transforming growth factor beta specically enhances IgA production by lipopolysaccharide-stimulated murine
B lymphocytes. J Immunol 2009;182:813.
Gandhi R, Farez MF, Wang Y, Kozoriz D, Quintana FJ, Weiner HL. Cutting
edge: human latency-associated peptide T cells: a novel regulatory T cell
subset. J Immunol 2010;184:46204.
rcamo CV, Hall J, Sun CM, Belkaid Y,
Coombes JL, Siddiqui KR, Arancibia-Ca
et al. A functionally specialized population of mucosal CD103 DCs induces
Foxp3 regulatory T cells via a TGF-beta and retinoic acid-dependent
mechanism. J Exp Med 2007;204:175764.

Please cite this article in press as: Medeiros SR, et al., Vitamin A supplementation leads to increases in regulatory CD4Foxp3LAP T cells
in mice, Nutrition (2015), http://dx.doi.org/10.1016/j.nut.2015.03.004