lable at ScienceDirect

Nutrition xxx (2015) 1–6

Contents lists avai

Nutrition

journal homepage: www.nutr i t ionjrnl .com

Basic nutritional investigation

Vitamin A supplementation leads to increases in regulatoryCD4þFoxp3þLAPþ 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,*aDepartamento de Bioqu�ımica e Imunologia, Instituto de Cie

ˇ

ncias Biol�ogicas, Universidade Federal de Minas Gerais, Belo Horizonte, BrazilbDepartamento de Morfologia, Instituto de Cie

ˇ

ncias Biol�ogicas, Universidade Federal de Minas Gerais, Belo Horizonte, BrazilcDepartamento de Nutric~ao, Escola de Enfermagem, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

a r t i c l e i n f o

Article history:Received 14 January 2015Accepted 18 March 2015

Keywords:Vitamin ARetinoic acidRegulatory T cellsTGF-b

This study was financially supported by scholarshipfrom Conselho Nacional de Desenvolvimento Cient�ı(SRM, AMF, DCC, NPR, FLG, and ACA), Coordenac~aoPessoal de N�ıvel Superior (CAPES) (LL and AGP), anPesquisa do Estado de Minas Gerais (FAPEMIG) (AFS)* Corresponding author. Tel.: þ55 313 499 2640; fa

E-mail address: afaria@icb.ufmg.br (A. M. C. Faria)

http://dx.doi.org/10.1016/j.nut.2015.03.0040899-9007/� 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Medeiros Sin mice, Nutrition (2015), http://dx.doi.org/1

a b s t r a c t

Dietary compounds, including micronutrients such as vitamin A and its metabolite retinoic acid,directly influence the development and function of the immune system. In this study, we showthat either dietary deficiency of or supplementation with vitamin A had immunologic effects inmice that were fed these diets during their development (for 8 wk during the postweaning period).Deficient mice presented higher levels of interferon-g, interleukin (IL)-6, transforming growthfactor-b, IL-17, and IL-10 in the gut-associated lymphoid tissues and draining lymph nodes, indi-cating a proinflammatory shift in the gut mucosa. Serum immunoglobulin G levels also wereelevated in these mice. Conversely, supplemented mice showed higher frequencies ofCD4þFoxp3þLAPþ regulatory T cells in gut lymphoid tissues and spleen, suggesting that vitamin Asupplementation in the diet may be beneficial in pathologic situations such as inflammatory boweldiseases.

� 2015 Elsevier Inc. All rights reserved.

Introduction

Dietary compounds directly influence the development andthe function of the immune system. Micronutrients, such as vi-tamins, have been described as important factors that affect bothinnate and adaptive immune responses. This is the case ofvitamin A, which is consumed in the form of retinol or b-caro-tene and metabolized to retinoic acid (RA), the active form of thenutrient [1].

Despite being found in many kinds of food, vitamin A defi-ciency is a global health problem [2]. Vitamin A deficiency mayresult in higher infant mortality, poor body development, and anincreased susceptibility to infections. These alterations are

s and research fellowshipsfico e Tecnol�ogico (CNPq)

de Aperfeicoamento ded Fundac~ao de Amparo �a, Brazil.x: þ 55 313 409 2613..

R, et al., Vitamin A supplem0.1016/j.nut.2015.03.004

mostly due to its key role in epithelial cell differentiation andintegrity. The immune system is also affected by vitamin Abecause RA is involved in switching immune response profilefrom T helper (Th)1 to Th2, reducing Th17 differentiation in vitro,in the differentiation of mucosal CD4þCD25þFoxp3þ Tregs, andin immunoglobulin (Ig)A production in the gut mucosa [3–5]. RAalso imprints gut-homing properties to activated Tand B cells [6].

The aim of this study was to analyze the clinical, biochemical,and immunologic effects brought about by either dietary defi-ciency of or dietary supplementation with vitamin A in micefrom weaning to adulthood.

Materials and methods

Animals, experimental design, and diets

Female C57 BL/6 mice were obtained from Centro de Bioterismo do Institutode Cie

ˇ

ncias Biol�ogicas (CeBio–ICB), Universidade Federal de Minas Gerais at day21 after birth. Animals were randomly divided in three groups that were fed dietscontaining different amounts of vitamin A: no vitamin A (VitA-deficient group;n ¼ 5), 4000 IU (control group; n ¼ 6), 10 000 IU (VitA-supplemented group;

entation leads to increases in regulatory CD4þFoxp3þLAPþ Tcells

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Fig. 1. Effect of vitamin A deficiency or supplementation in weight gain. After weaning (day 21 after birth), mice were fed either vitamin A-deficient or vitamin A-sup-plemented 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 liquidchromatography in liver and serum. Bars represent the mean � SEM of a group (n ¼ 6). (B) Weekly weight measurement of mice were performed and dots represent themean þ SD of a group (n ¼ 6). Differences among groups were calculated using student’s t test, and statistical significance is represented by * (P < 0.05), y(P < 0.001), andz(P < 0.0001).

S. R. Medeiros et al. / Nutrition xxx (2015) 1–62

n¼ 7). All mice were fed AIN93 G diet (modified or not) for 8 wk fromweaning toadulthood (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 in-testine harvesting, colonic tissue was placed in buffer solution with proteaseinhibitor (1 mL/0.1 g). Tissue fragments were homogenized and centrifuged andsupernatants were collected for cytokine assay. Interleukin (IL)-10, IL-6, trans-forming growth factor (TGF)-b, interferon (IFN)-g and IL-17 were measured byenzyme-linked immunosorbent assay (ELISA) as previously described [8]. Allreagents were obtained from BD-Pharmingen (Franklin Lakes, NJ, USA).

Histopathologic analysis

Colon was fixed in 10% formalin in 0.01 M phosphate buffer (pH 7.2) andembedded in paraffin. Sections were stained with hematoxylin and eosin forhistopathologic examination by light microscopy as described previously [9]. Forhistologic analysis, the mucosal architecture, muscle thickening, goblet celldepletion, crypt abscesses, and infiltrating cell counts were evaluated and pho-tographed using a digital camera (Moticam 2500, China) coupled with an opticalmicroscope (Olympus Optical Co., Japan).

Collection of sera, intestinal lavage fluid, and immunoglobulin measurement

Blood samples were collected from the axillary plexus in mice under anes-thesia, and sera separated. Intestinal lavage fluids were collected after euthanasiaflushing the entire intestine with cold-buffered saline (phosphate-buffered sa-line). Serum Igs (IgG, IgM, and IgA) as well as secretory IgA in the intestinal fluidwere measured by sandwich ELISA as previously described [8] using antibodiesfrom Southern Biotechnology (Birmingham, AL, USA).

Cell preparation and flow cytometry analysis

Cells of lamina propria were isolated by a modified version of a previouslyreported method [10], as described elsewhere [8]. Spleen and mesenteric lymphnodes (MLN) cells were also isolated. Cells from lamina propria compartments,MLN, and spleen were surface labeled with Fluorescein isothiocyanate-conjugated antimouse CD4, a combination of biotin antimouse latency-associated peptide (LAP) and allophycocyanin-conjugated streptavidin andintracellularly with phycoerythrin-conjugated antimouse Foxp3 (BD Pharmin-gen). Labeled cells were acquired using FACScalibur (Becton Dickinson, MountainView, CA, USA). At least 30 000 events were counted and data were analyzedusing Flow Jo software.

High-pressure liquid chromatography

For high-pressure liquid chromatography assay, a C18 columnwas used withacetonitrile/dichloromethane/methanol (70/20/10) as mobile phase. Plasma andliver samples were treated with ethanol and n-hexane and dried under nitrogenas previously described [11].

Please cite this article in press as: Medeiros SR, et al., Vitamin A supplemin mice, Nutrition (2015), http://dx.doi.org/10.1016/j.nut.2015.03.004

Statistical analyses

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

Results

Vitamin A deficiency does not induce changes in body weight

To evaluate the amount of vitamin A absorbed under eitherdeficiency or supplementation dietary conditions, the concen-tration of vitamin A was measured in both the serum and in theliver. As expected, the storage levels of vitamin A in the liver ofdeficient mice were very low, and levels in the supplementedanimals were higher compared with control mice. Serum levelsof vitamin A in animals from control and supplemented groupswere similar, although a reduction was observed in the deficientgroup (Fig. 1A).

To study clinical changes that vitamin A deficiency and sup-plementation could bring about, animal’s weight was verifiedweekly during the 8-wk experimental period. There was nosignificant difference in the weight gain among the animals(Fig. 1B). There was no difference in food intake during thewholeexperimental period either (data not shown). Because vitamin Alevels were altered in the livers of supplemented and deficientmice, we evaluated basic biochemical parameters to examinewhether those alterations lead to disturbances in serumcholesterol, triacylglycerols, and glucose levels as well as in liverlipid concentrations, but no differencewas observed in any of theparameters evaluated (data not shown).

Vitamin A deficiency did not affect mucosal architecture

It is well known that vitamin A is important to maintain theintegrity of intestinal epithelia [12]. We performed colon histo-logic analysis to study whether 8-wk vitamin A deficiency wouldlead to morphologic changes in the colon mucosa. No alterationwas found in mice that were fed vitamin A-deficient or vitaminA-supplemented diets (Fig. 2). The thickness of muscle layer ormucosa muscular was similar in all groups. The submucosal wasthin with no signs of either edema or inflammatory infiltratedcells. All groups showedmucosawith globet cells, intestinal cells,and absence of crypt abscesses.

entation leads to increases in regulatory CD4þFoxp3þLAPþ Tcells

Fig. 2. Morphologic study of the colonic mucosa of mice fed either vitamin A-deficient or vitamin A-supplemented diets. Colonic tissues were collected, fixed in bufferedsaline, 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 infiltrate in the submucosal layer, thickening of muscle layer, and presence of crypt abscesses. (A, B)control group; (C, D) vitamin A-deficient 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.

S. R. Medeiros et al. / Nutrition xxx (2015) 1–6 3

Vitamin A deficiency led to elevated serum IgG levels andincreased cytokine production at mucosal sites

To evaluate the effect of vitamin A in immune cell function,serum and secretory Ig levels as well as levels of cytokines in gut-associated lymphoid tissues were measured. Serum IgG levelswere elevated in vitamin A-deficient mice but unaltered in sup-plemented mice compared with control animals (Fig. 3A). Secre-tory IgA (Fig. 3A) as well as serum IgM and IgA levels were notaffect by vitamin A deficiency or supplementation (data notshown). Production of IFN-g, IL-17, IL-6, TGF-b, and IL-10 (Fig. 3B–F) was increased in the colonic tissues of deficient mice, IFN-g(Fig. 3B) and IL-6 (Fig. 3D) levels were also increased in MLN ofvitamin A-deficient mice. A reduction was observed in the pro-duction of IL-17 in the MLN of the supplemented group (Fig. 3C).Noalterations in cytokineproductionweredetected in the spleen.

Please cite this article in press as: Medeiros SR, et al., Vitamin A supplemin mice, Nutrition (2015), http://dx.doi.org/10.1016/j.nut.2015.03.004

Vitamin A supplementation led to increased frequencies ofCD4þFoxp3þLAPþ regulatory T cells

Because vitamin A is involved in the differentiation of Foxp3þregulatory T (Tregs) cells in the gutmucosa, we next analyzed thefrequency of Tregs expressing Foxp3 and the surface form of TGF-b (LAPþ cells). There was an increase in the frequency ofCD4þFoxp3þLAPþ cells in spleen, MLN, Peyer’s patches, andcolon lamina propria of vitamin A-supplemented mice comparedwith both control and deficient mice (Fig. 4).

Discussion

In this study, we examined the clinical, biochemical, andimmunologic effects of either dietary deficiency of or supple-mentation with vitamin A in mice during their development (for

entation leads to increases in regulatory CD4þFoxp3þLAPþ Tcells

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Fig. 3. Effect of vitamin A deficiency and supplementation on Ig secretion and cytokine production in gut-associated lymphoid tissues. Serum Igs and secretory IgA in theintestinal fluid (A) was measured by sandwich ELISA. MLN, colonic tissues, and spleen were collected and homogenized in buffered saline with protease inhibitors. Cytokinesin the supernatants of tissue homogenates (B–F) were measured by ELISA. Bars represent mean þ SEM of a group (n ¼ 7). Differences among groups were calculated usingANOVA, and statistical significance 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.

S. R. Medeiros et al. / Nutrition xxx (2015) 1–64

8 wk in the postweaning period). Initially, we examined the ef-fects of dietary manipulation in serum and liver storage levels ofthe hydrophobic vitamin. All mice, including those that were feda vitamin A-deficient diet after weaning, received a supply ofvitamin A by breast-feeding because mother’s milk is one of themajor sources of vitamin A. This is probably why they had asignificant stock of this vitamin in the liver at the beginning ofthe experiment. However, 8 wk of insufficient ingestion ofvitamin A was enough to lower the level of this nutrient in liverand serum. Despite that, none of the dietary manipulationsinduced changes in weight gain. Vitamin A is important for theepithelial growth and maintenance [12], but the period ofdeprivation was not enough to induce histologically detectabledisturbances in the gut mucosa (Fig. 2). The only change noticedwas a slight augmentation in the size of epithelial-related gobletcells in supplemented mice (Fig. 2C).

Although dietary modifications in vitamin A content did notlead to changes in the gut mucosa architecture, some immunefunctions were altered mostly in the gut-associated lymphoidtissue and draining lymph nodes. All cytokines measured (IFN-g,IL-6, IL-17, TGF-b, and IL-10) were increased in the colon ofvitamin A-deficient mice. IFN-g and IL-6 were also increased inthe MLN of these mice. Cytokine IL-6, together with TGF-b, hasan important role in driving the differentiation of Th17 cells [6,13]. As both cytokines were increased in the colon, it is plau-sible that the higher levels of IL-17 were due to an enhancedgeneration of Th17 cells. Levels of the anti-inflammatory cyto-kine IL-10 were also increased in the colon, suggesting acompensatory loop provided by the local inflammatory condi-tions as previously shown in experimental models of colitis [14]

Please cite this article in press as: Medeiros SR, et al., Vitamin A supplemin mice, Nutrition (2015), http://dx.doi.org/10.1016/j.nut.2015.03.004

and asthma [15]. Along with this proinflammatory profile ofcytokine production, serum IgG levels were elevated in deficientmice. IgG production is dependent on IFN-g secretion [16] by Tcells and, although there is no change in the levels of this cyto-kine in spleens of deficient mice, it is possible that the higherlevels of IFN-g in mucosal associated organs had an effect in thesystemic levels of this immunoglobulin. Despite the alteration inthe cytokine profile in the gut mucosal tissues, it did not influ-ence secretory IgA production. Levels of TGF-b were higher indeficientmice and this cytokine is critical for IgA production [17],therefore the increase in inflammatory cytokines may have notbeen enough to interfere with secretory IgA secretion in the gut.

On the other hand, supplementation with vitamin A led to anincrease in the frequency of a subpopulation of Treg cellexpressing a membrane bound form of TGF-b associated withLAP [18]. It was already shown that RA, a metabolite of vitamin A,can be produced by CD103þ dendritic cells expressing theenzyme RALDH in the intestinal mucosa. RA acts along with TGF-b in the differentiation of naïve CD4þT cells into regulatoryCD4þFoxp3þT cells in vitro [5] and in vivo [19]. To our knowl-edge, this study demonstrated for the first time that supple-mentation of this nutrient in the diet for 8 wk could increase thepopulation of regulatory CD4þFoxp3þLAPþT lymphocytes in allorgans analyzed, but no difference was observed in the wholepopulation of CD4þFoxp3þT cells. It is possible that in fullydeveloped mice at physiological conditions, CD4þFoxp3þTregsare already at their optimal frequencies. However,CD4þFoxp3þLAPþT cells could represent a subset amongFoxp3þTregs that are more sensitive to the effects of vitamin A,and therefore were the one affected by its increased levels.

entation leads to increases in regulatory CD4þFoxp3þLAPþ Tcells

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Fig. 4. Effect of vitamin A on the frequencies of regulatory CD4þFoxp3þLAPþ T cells. Cells from colonic lamina propria, MLN, Peyer’s patches, and spleen were isolated,stained with fluorochrome-labeled antibodies and analyzed by flow cytometry. Frequencies of CD4þFoxp3þLAPþ cells in spleen (B), MLN (C), Peyer’s patches (D), and colonlamina 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 ¼ 4–7). Differences among groups were calculated using ANOVA test. Significant differences are represented by * (P < 0.05), y(P < 0.01), and z(P < 0.0001). ANOVA, analysis ofvariance; LAP, latency-associated peptide; MLN, mesenteric lymph node.

S. R. Medeiros et al. / Nutrition xxx (2015) 1–6 5

Conclusion

Vitamin A is essential to themaintenance of the homeostasis ofgut lymphoid cells. In this study, its deficiency led to disturbancesin inflammatory cytokines in the gutmucosa aswell as in systemicIg production. Conversely, to our knowledge, we showed for thefirst time, that vitamin A supplementation of even healthy miceboosted the differentiation of CD4þFoxp3þLAPþTregs in localand systemic lymphoid organs. Therefore, this dietary manipula-tion may help to boost Treg responses in pathologic conditionssuch as inflammatory bowel diseases.

Please cite this article in press as: Medeiros SR, et al., Vitamin A supplemin mice, Nutrition (2015), http://dx.doi.org/10.1016/j.nut.2015.03.004

Acknowledgment

The authors acknowledge Ilda Marcal de Sousa for theexcellent care of the mice.

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