Nutrition 30 (2014) 350357

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Nutrition
journal homepage: www.nutritionjrnl.com

Basic nutritional investigation

Gestational vitamin A deciency reduces the intestinal

immune response by decreasing the number of immune cells
in rat offspring
Xia Liu M.D. a, Yingying Li M.D. a, Yuting Wang M.D. a, Qinghong Wang Ph.D. b,
Xin Li M.D. b, Yang Bi Ph.D. a, Lan Liu M.D. c, Xiaoping Wei M.D. a, Tingyu Li M.D. a,
Jie Chen Ph.D. a, *
a
Children Nutrition Research Center, Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in
Chongqing, CSTC2009 CA5002, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders,
Childrens Hospital of Chongqing Medical University, Chongqing, P.R. China
b
Institute of Pediatrics, Childrens Hospital of Chongqing Medical University, Chongqing, P.R. China
c
Laboratory of Intestinal Physiology and Pathology, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 4 February 2013
Accepted 17 September 2013

Objectives: Vitamin A (VA) is a critical micronutrient for life, especially during growth and development. There is a close relationship between VA deciency (VAD) and the morbidity of diarrhea
in the clinical setting. However, the regulatory mechanisms of VA are not clearly understood.
Methods: Specic-pathogenfree Wistar rats received a diet with or without VA before gestation.
The offspring were submitted to an abdominal injection of Escherichia coli lipopolysaccharide. After
the challenge, which lasted for 12 h, the serum retinol was detected by high-performance liquid
chromatography, and the level of immunoglobulin A in the stool was analyzed by enzyme-linked
immunosorbent assay. The lymphocyte immunophenotypes were evaluated with the use of ow
cytometry with samples collected from the spleen, the mesenteric lymph nodes, Peyer patches,
and intestinal intraepithelial lymphocytes.
Results: Early life VAD, independent of the lipopolysaccharide challenge, signicantly decreased
serum retinol level and CD8 intestinal intraepithelial lymphocytes. The level of immunoglobulin A
secretion and percentages of splenic CD4CD8 T cells were affected by the interaction effects of
the lipopolysaccharide challenge and VAD treatment. Gestational VAD signicantly increased the
percentages of B cells in the mesenteric lymph nodes and decreased the percentages of CD11 C
dendritic cells and CD4CD25 T cells from the Peyer patches. The lipopolysaccharide challenge
only signicantly increased percentages of splenic CD4CD25 T cells. The intestinal tissue of the
pups with VAD displayed mild inammation.
Conclusions: Gestational or early life VAD decreases the numbers of immune cells in offspring,
which may partly suppress the activities of the mucosal immune responses in the intestine. This
suggests that more attention should be given to the VA nutritional state of children and women of
reproductive age.
2014 Elsevier Inc. All rights reserved.

Keywords:
Vitamin A deciency
Gestation
Early life period
Lymphocytes
Lipopolysaccharides

Introduction
Vitamin A (VA) and its metabolites play a critical role in
development during the gestational and the early postnatal
X.L. and Y.L. conducted the experiment and drafted the manuscript. Y.W. and
X.W. established the animal model. Q.W. and X.L. detected the lymphocytes using
ow cytometry. Y.B., L.L., and T.L. helped to design the study. J.C. provided funding
and overall direction. All authors read and approved the nal manuscript.
* Corresponding author. Tel.: 86-23-63630913; fax: 86-23-63622754.
E-mail address: jchen010@gmail.com (J. Chen).
0899-9007/$ - see front matter 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nut.2013.09.008

period [1]. VA deciency (VAD) is known to be a major public

health and nutrition problem worldwide, especially in developing countries. VAD can limit growth, cause blindness, weaken
innate and acquired host defenses, exacerbate infection,
and increase the risk of death. Our previous studies have
demonstrated that supplementation with VA or with VA in
combination with multiple micronutrients can improve the
physical growth levels [2], anemia status [3], and infection
morbidity of preschool children [4]. Many studies have suggested
that a lower nutritional level of VA is correlated directly with

X. Liu et al. / Nutrition 30 (2014) 350357

the pathogenesis of diarrhea [57]. However, few studies have

been conducted on the effect of VAD on the mucosal immune
response of the intestine.
Increasing evidence has revealed that VAD decreases the intestinal barrier function, thereby increasing the intestines susceptibility to infection by pathogens, which may result in
diarrhea [6,8]. Supplementation with the appropriate amount of
VA acts as an anti-infection agent, which reduces infant mortality rate [9] and the morbidity associated with diarrhea [10].
Several studies have demonstrated the roles of VA and retinoic
acid (RA) receptors in T-cell differentiation and of immunoglobulin A (IgA) switching and production [1115]. However, the
regulatory functions of VA in the intestinal mucosa are unknown
(especially Peyer patches and intestinal intraepithelial lymphocytes [IELs]). Few researchers have studied the effects of VAD
from the beginning of gestation on the postnatal intestinal
mucosal immunity.
A rat model of VAD during the gestational period was used
in the present study to investigate whether gestational VAD
affected immune activity and impaired the capacity of the immune response in offspring after the challenge. We rst observed
the differences in the intestinal IgA levels and the pathologic
changes of the intestinal mucosa between the VAD and VA
normal (VAN) pups. The percentage changes in the various
lymphocyte subsets were analyzed in the spleens of the VAD
offspring after lipopolysaccharide (LPS) challenge. Second, we
focused on the study of gut-associated lymphocyte tissues
(GALT)dincluding the mesenteric lymph nodes (MLNs), Peyer
patches (PPs), and IELsdto investigate the immunomodulatory
mucosal immune response of the intestine. Third, we used the
pups of the VAD gestational rats that were fed with the maternal
milk of VAN rats to elucidate the importance of the VA nutritional level during the gestational period for postnatal immune
defense. This study provides a new insight into the role that
VAD during pregnancy plays in the immunosuppression of the
mucosal immune response in the intestine during postnatal
development.
Materials and methods
Animals, diets, and the lipopolysaccharide challenge
The use and reference numbers of the animals were approved by the Animal
Experimentation Ethical Committee of Chongqing Medical University (Chongqing, China). Forty 6-wk-old female specic-pathogenfree Wistar rats obtained
from the Experimental Animals Center of the Third Military Medical University
(Chongqing, China) were randomly selected and divided equally into two groups
of maternal VAN and maternal VAD. The maternal VAD rats were fed a VAD diet
that contained 400 IU/kg of VA for 4 wk to construct the VAD animal model
before gestation, whereas the VAN rats received VAN food that contained 6500
IU/kg of VA as the control [16,17]. When the serum retinol levels of blood samples
taken from the tails of the VAD rats decreased to 1.05 mmol/L, the 20 maternal
VAD rats were mated with normal males. The pregnant maternal rats were fed
either the VAD diet or the VAN diet during both the gestational and lactation
periods to maintain the retinol levels in the serum. All rats were housed at a
constant temperature of 22 C to 24 C with 60% relative humidity, with articial
daylight from 07:00 to 19:00 every day and with VAD or VAN food and water
available ad libitum. The specic-pathogenfree animal house was certied for
experimental animals. Each dam nursed eight pups from her litter during the
lactation period. After weaning, the eight pups were randomly separated into
two cages.
After a weaning period of 21 postnatal days, the pups were fed continuously
for 3 wk with the VAD diet and designated as the VAD group (n 60) or the VAN
diet and designated as the VAN group (n 60). After birth, the VAD pups were
cross-fostered to VAN dams to make the VAD-N pups (n 60) during the
lactation period and then fed the VAN diet after weaning (Fig. 1). Approximately
half of the total pups in each group were randomly selected to be injected
abdominally with Escherichia coli LPS (i.e., 3 mg of LPS per kilogram of animal
weight). The other half of the pups were injected with approximately 0.25 to

351

Fig. 1. Schematic diagram showing the three offspring groups: VAN, VAD, and VADN. The VAN pups were delivered and nursed by maternal VAN rats and then fed a
VAN diet for 3 wk after weaning. The VAD pups were birthed and nursed by
maternal VAD rats and fed a VAD diet for 3 wk after weaning. The VAD-N pups
were delivered from maternal VAD rats and then nursed by maternal VAN rats and
fed with a VAN diet until they were 6 wk old. VAD, vitamin A deciency; VAD-N,
VAD during gestation and fed the milk of VAN rats after birth; VAN, vitamin A
normal.
0.4 mL (i.e., about the same volume as the LPS injection) of phosphate buffered
saline (PBS) to serve as the sham group for the LPS challenge. After the LPS
challenge had lasted 12 h, the 6-wk-old pups were anesthetized with chloral
hydrate, and the blood was collected immediately from the femoral artery. The
isolated tissues of the spleen, the MLN, and the intestine were extracted and
placed into PBS until they were used in the following steps.
Detection of serum retinol
The concentration of serum retinol was determined with the use of highperformance liquid chromatography according to our previously described
methods [18] with slight modications. Briey, 200 mL of serum was deproteinized with dehydrated alcohol, and then the retinol was extracted with hexane
and evaporated with nitrogen gas. The residue was dissolved in 100 mL of the
mobile phase mixture (methanol:water ratio of 97:3), and the entire sample was
transferred to a bottle installed in the high-performance liquid chromatography
apparatus (DGU-20 As, Shimadzu Corporation, Japan). The retinoids were separated by chromatography on an analytical column (Hypersil phenyl 120 A 5 mm,
250  4.6 mm, Phenomenex, USA) via gradient elution of the mobile phase in a
liquid chromatograph equipped with a 315 nm ultraviolet photodiode array
detector.
Hematoxylin and eosin staining and enzyme-linked immunosorbent assay
measurements
After the samples of fresh intestine were xed in 4% paraformaldehyde,
dehydrated, embedded in parafn, and sectioned in accordance with standard
methods, the serial sections, which were 4-mm thick, were stained with hematoxylin and eosin to be examined under a light microscope for pathologic
observation.
As described for the reported method of Frossard et al. [19], 100 mg of feces
from the ileocecum were resuspended in 1 mL of 0.01 M PBS that contained 1%
fetal bovine serum (FBS) and 0.1 mM phenylmethanesulfonyl uoride (Solarbio,
Amresco). The concentration of total IgA in the supernatants was measured with
the use of an enzyme-linked immunosorbent assay kit (Bethyl Lab, Inc, Montgomery, Texas, US) according to the manufacturers instructions.
Isolation of lymphocytes from the spleen and the mesenteric lymph nodes
The spleens and the MLNs of the rats were cut into pieces with the use of
surgical scissors on a 200-mesh metallic grid and then washed with PBS. The
individual lymphocytes were collected via centrifugation. After washing, 3  107
cells were resuspended in PBS for analysis.
Peyer patch preparation
Lymphocytes from PPs were isolated as described previously [20]. Briey, the
lymphoid follicles of PPs were carefully excised from the intestine. Single lymphocytes were released with 2 mL of FBS after disruption with the use of surgical
scissors on a 300-mesh metallic grid. The lymphocyte suspension was slowly
added onto the surface of a lymphocyte separation medium for rats (Tianjinghaoyang Biological Manufacture Co., China) and centrifuged at 805 g for 15 min.
The white occulent at the interface between the FBS and the lymphocyte separation medium was collected into a new tube with a Pasteur pipette, washed
twice, and then kept in 100 mL of PBS for ow cytometry.

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X. Liu et al. / Nutrition 30 (2014) 350357

Fig. 2. Effects of VAD on the levels of serum retinol, IgA secretion, and the histopathologic changes of intestinal mucosa in 6-wk-old pups. (A) The changes in the serum
retinol levels (mmol/L) among the VAN, VAD, and VAD-N groups after the presence or absence of Escherichia coli LPS challenge as assessed by HPLC (n 15). (B) The VA main
effect, independent of LPS treatment, as shown by the serum retinol levels (mmol/L) among the combined VAN, VAD, and VAD-N groups. (C) The levels of IgA in the intestinal
stool among the VAN, VAD, and VAD-N groups with or without LPS treatment (n 6). (D) Histologic examination of intestinal mucosa staining with hematoxylin and eosin (n
3). Interaction indicates an effect of LPS treatment versus null treatment. * P < 0.05; y P < 0.01; z P < 0.001; n.s. not signicant in post hoc tests. HPLC, high performance
liquid chromatography; IgA, immunoglobulin A; LPS, lipopolysaccharide; VAD, vitamin A deciency; VAD-N, VAD during gestation and fed the milk of VAN rats after birth;
VAN, vitamin A normal.
Isolation of intestinal intraepithelial lymphocytes
With the use of certain modications that have been described previously
[21], the entire intestine was washed with 0.01 M PBS three times to remove the
intestinal contents and mucus. The intestine was then incubated in ice-cold PBS
that contained 2% FBS for 2 h; the whole intestine without the PPs was washed
twice with warmed (37 C) Dulbeccos modied Eagles medium with 2% FBS and
1% dithiothreitol and then gently squeezed with ophthalmic tweezers from the
serosal side to remove the loosened cells on the intestinal mucosal surface. The
cellmedium mixture was placed at room temperature for 10 min, and the supernatant was collected into a new tube for centrifugation. The pellet was
resuspended in FBS, and the lymphocytes were isolated with lymphocyte separation medium for detection with the use of ow cytometry.
Flow cytometric analysis
The immunophenotypes of the various lymphocytes were used to identify the
specic cell types with the following antibodies (BD Pharmingen, Heidelberg). The
T lymphocytes were stained with PECy5-anti-CD45, APC-anti-CD3, PE-anti-CD4,
and FITC-anti-CD8. The B lymphocytes were stained with PECy5-anti-CD45 and
FITC-anti-CD45 R, whereas the dendritic cells (DCs) were stained with PECy5-antiCD45 and PE-anti-CD11 c. The CD4CD25 T cells were analyzed with APC-antiCD3, PE-anti-CD4, and FITC-anti-CD25. The IELs were stained with APC-anti-CD3,
FITC-anti-CD8, and PE-anti-TCRgd. The corresponding isotype-matched monoclonal antibodies were used as negative controls. The percentages of the various
lymphocytes were determined with a BD FACSCanto II ow cytometer (Becton
Dickinson).
Statistics
All data were expressed as mean  SD. Signicant differences were calculated
via two-way analysis of variance with the Bonferroni post hoc test with the use of
the GraphPad Prism version 5.0 software package. Statistically signicant interactions were analyzed with the Bonferroni post hoc test between LPS and noLPS treatments. When there was no signicant interaction, the main effects were
analyzed with the use of the Tukey post hoc test for the three combined VA
treatment groups, and the Student t test was used for the two combined LPS
treatment groups. Only the relevant comparisons of the combined groups are

presented in the Results section [22,23]. P values of <0.05 were accepted as

statistically signicant.

Results
Lower levels of serum retinols in the early life vitamin Adecient
offspring
We induced the VAD female model before pregnancy in
accordance with the international standard for VA levels in
humans [24]. The level of serum retinol in the VAD pups (0.641
[0.184] mmol/L) was signicantly lower than that of the VAN pups
(1.471 [0.337] mmol/L) (P < 0.001) (Fig. 2A). Among the VAD-N
pups, serum retinol levels signicantly increased (1.45 [0.46]
mmol/L) to the levels of VAN pups in the absence of LPS treatment
(Fig. 2A). The Tukey post hoc analysis of the three combined VA
treatment groups showed that the retinol levels in the combined
VAD pups were signicantly decreased as compared with those in
both the combined VAN and VAD-N groups (Fig. 2B). Although
the concentration of retinol in the VAD-N group (1.037 [0.451]
mmol/L) was slightly lower than that found in the VAN group after
the LPS challenge (Fig. 2A), there was no statistical difference in
both of the combined groups without or with LPS treatment (data
not shown). These data demonstrated that VAD during the early
life period decreased the retinol level of the pups.
Reduction of intestinal immunoglobulin A and local inammatory
changes after the lipopolysaccharide challenge in the vitamin A
decient pups
Secretory IgA is the rst line of humoral immune defense at
the intestinal mucosal surface that provides protection against

X. Liu et al. / Nutrition 30 (2014) 350357

353

Fig. 3. The percentage of (A) CD4CD25 T cells and (C) CD4CD8 double-positive T lymphocytes in the spleen of VAN, VAD, and VAD-N groups with or without LPS
challenge assessed with the use of ow cytometry (n 8). (B) The LPS main effect, independent of VA treatment, in the percentage of CD4CD25 T cells in the spleen.
Interaction indicates an effect of LPS treatment versus null treatment. *P < 0.05, yP < 0.01; zP < 0.001; n.s. not signicant in post hoc tests. LPS, lipopolysaccharide; VAD,
vitamin A deciency; VAD-N, VAD during gestation and fed the milk of VAN rats after birth; VAN, vitamin A normal.

pathogen invasion. The levels of IgA in the intestinal fecal material were not signicantly different in the offspring among the
VAN, VAD, and VAD-N pups treated without LPS (Fig. 2C). The IgA
level in the VAN pups after the LPS challenge sharply increased to
159.3 (69.28) ng/mL, which was a 10-fold increase as compared
with the VAN rats that were not treated with LPS (P < 0.001).
However, the LPS challenge only caused a 2.88-fold increase in
the IgA levels in the VADLPS pups and a 5.41-fold increase in
the VAD-NLPS pups. These values were signicantly different
when compared with the VANLPS group (P < 0.001 and P <
0.01, respectively) (Fig. 2C). The Bonferroni post hoc analysis of
interaction between LPS treatment and null treatment showed
that the P value of the interaction was <0.0001. These results
indicated that the LPS challenge increased the IgA levels and that
VAD during gestation suppressed the intestinal IgA secretion
induced by the LPS treatment, which may lead to antiinammatory dysfunction during a response to an invading
pathogen.
Histopathologic observation demonstrated that the villi of the
intestines in the VAD and VAD-N pups displayed slight edema
and recruited more enlarged goblet cells in the intestinal
epithelium layer. The LPS challenge caused more serious
inammation in the lamina propria and the intestinal mucosa in
both VAD and VAD-N pups (Fig. 2D).
Effects on the splenic T-lymphocyte subsets in offspring during
early life vitamin A deciency
To evaluate the effects of VAD during the gestational period
on the immune response of pups with or without LPS challenge,
ow cytometry was used to analyze the subsets of lymphocytes
present in the spleen. The data shown in Figure 3A demonstrate
that there were signicant main effects of LPS treatment (P <
0.0001) and also of VA treatment (P 0.0024) in a percentage of
CD4CD25 T cells. However, we found that the ratio of
CD4CD25 T cells in the combined LPS treatment group was
signicantly increased as compared with the combined no-LPS
challenge group (Fig. 3B). However, there were no signicant
differences in the number changes of CD4CD25 T cells among
the combined VAN, VAD, and VAD-N groups (data not shown).
These ndings demonstrated that LPS treatment effectively
increased the number of CD4CD25 T cells in the spleen.
Meanwhile, we were surprised to nd that the percentage of
CD4CD8 T cells dropped to 8.0% (1.8%) in the VAD pups as
compared with 14.1% (3.5%) in the VAN pups in the absence of

LPS challenge (P < 0.001) (Fig. 3C). The Bonferroni post hoc
analysis of the interaction between LPS and no-LPS treatments
showed that the percentage of CD4CD8 T cells was signicantly repressed by the LPS exposure in both the VAD and VAN
pups as compared with the LPS null treatment (P < 0.001 and P <
0.01, respectively) (Fig. 3C). Furthermore, the number of
CD4CD8 T cells in the VAD-N pups was the same as that of the
VAD pups. However, no differences in the percentage of
CD4CD8 T cells among the three groups after LPS treatment
were found (Fig. 3C). These ndings suggest that gestational VAD
decreased the number of CD4CD8 double-positive (DP) T
lymphocytes in the absence of LPS treatment.
Increased percentage of B lymphocytes in the mesenteric lymph
nodes of vitamin Adecient pups
The MLN is part of the gut-associated lymphoid tissue, and it
acts as a link between the systemic immune response and the
mucosal immune response of the intestine. The subsets of lymphocytes in the MLN were observed in this study, and we found
that the percentage of B lymphocytes in the MLN of the VAD
pups was slightly increased as compared with the VAN pups with
or without LPS challenge. The numbers of B cells in the VAD-N
group were similar to those of the VAD group in the presence
or absence of LPS treatment (Fig. 4A). The two-way analysis of
variance showed that VA treatment was the main factor that
increased the number of B cells in the MLN (P < 0.0001). The
percentage of B cells in the combined VAN group was signicantly decreased as compared with the combined VAD and VADN groups (Fig. 4B), thereby suggesting that VAD during gestation
increased the percentage of B cells in the MLN. This pattern was
independent of LPS treatment.
Suppression of the mucosal immune response in the intestine as a
result of vitamin A deciency
As an extension of the ndings obtained from splenic and
MLN tissue, we further determined whether VAD during the
gestational period affected the development of the local intestinal immune cells, specically in the PPs and IELs. PPs are
specialized collective lymphoid nodes of the gut that have been
implicated as intestinal inductive sites during the mucosal immune response. The data shown in Figure 5A revealed that the
percentage of CD11 C DCs within the PPs was decreased in both
VAD and VAD-N pups as compared with the VAN pups with or

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X. Liu et al. / Nutrition 30 (2014) 350357

Fig. 4. Increase in the number of B lymphocytes in the MLN as a result of (A) gestational VAD (n 8 to 10) and (B) the VA signicant main effect, independent of LPS
treatment, on the percentage of B cells in the MLN among the three combined VAN, VAD, and VAD-N groups. Interaction indicates an effect of LPS treatment versus null
treatment. zP < 0.001; n.s. not signicant in post hoc tests. LPS, lipopolysaccharide; MLN, mesenteric lymph node; VAD, vitamin A deciency; VAD-N, VAD during gestation
and fed the milk of VAN rats after birth; VAN, vitamin A normal.

without LPS challenge. These differences represented the effects

of VA treatment (P < 0.0001) but not of LPS treatment (P
0.1075). After combining the two LPS treatments of the VAN,
VAD, and VAD-N groups, the percentages of the CD11 C DCs in
the combined VAD and VAD-N groups were signicantly
decreased as compared with those of the combined VAN pups
(Fig. 5B) The trend in the changes of the CD4CD25 T-cell subset
in the PPs was identical to that of the CD11 C DCs among the
three groups with or without LPS treatment (Fig. 5C, D); the P
values of the VA and LPS effects were <0.0001 and 0.0685,
respectively. These data demonstrated that gestational VAD

independent of LPS treatment decreased the number of CD11 C

DCs and CD4CD25 T cells in PPs. However, the CD4CD25 T
lymphocytes in the PPs were not induced by the LPS exposure in
the present study. The reason for this may have been that the LPS
challenge was administered for a short period via an abdominal
injection, which only resulted in systemic immune responses
and not in mucosal-based immunity of the intestine.
IELs reside within the intestinal epithelium to ensure the
integrity of the gut barrier function. They form one of the main
branches of the immune system, and they play a critical role in
the mucosal immune response of the intestine. Most IELs,

Fig. 5. Suppression of (A) CD11 C dendritic cells and (C) CD4CD25 T cells in PPs by gestational VAD in the presence or absence of LPS treatment (n 5 to 6). The VA
signicant main effect, independent of LPS treatment, on the percentage of (B) CD11 C dendritic cells and (D) CD4CD25 T cells in PPs among the three combined VAN, VAD,
and VAD-N groups. Interaction indicates an effect of LPS treatment versus null treatment. zP < 0.001; n.s. not signicant in post hoc tests. LPS, lipopolysaccharide; PPs,
Peyer patches; VAD, vitamin A deciency; VAD-N, VAD during gestation and fed the milk of VAN rats after birth; VAN, vitamin A normal.

X. Liu et al. / Nutrition 30 (2014) 350357

355

Fig. 6. Changes in the percentage of (A) CD8 and (C) CD8 TCRgd IELs among the pups of the VAN, VAD, and VAD-N groups in the presence or absence of LPS challenge (n
5 to 7). The VA signicant main effect, independent of LPS treatment, on the percentage of (B) CD8 and (D) CD8 TCRgd IELs among the three combined VAN, VAD and
VAD-N groups. Interaction indicates an effect of LPS treatment versus null treatment. *P < 0.05; yP < 0.01; zP < 0.001; n.s. not signicant in post hoc tests. IELs, intestinal
intraepithelial lymphocytes; LPS, lipopolysaccharide; TCR, T-cell antigen receptor; VAD, vitamin A deciency; VAD-N, VAD during gestation and fed the milk of VAN rats after
birth; VAN, vitamin A normal.

especially in the small intestine, constitutively express a hallmark of CD8 T cells and TCRgd [25]. As shown in Figure 6A, the
ratio of IELs with the CD8 phonotype was much lower in the
VAD pups as compared with the VAN pups; however, the CD8
IELs in the VAD-N group were similar to those of the VAN group
with or without LPS treatment. Under the VA main effect (P
0.0002) independent of LPS treatment, the number of CD8 IELs
in the combined VAD pups was signicantly decreased as
compared with those in the combined VAN and VAD-N pups
(Fig. 6B), thereby suggesting that VAD during early life but not
during gestation suppressed the percentage of CD8 IELs. The VA
treatment was also the main effect that impaired the percentage
of CD8TCRgd IELs (P 0.0006), whereas the LPS effect did not
(P 0.1046) (Fig. 6C). The numbers of CD8TCRgd IELs were
signicantly reduced in the combined VAD-N group as compared
with both the combined VAD and VAN groups (Fig. 6D). Taken
together, these results indicate that early life VAD could impair
the activity of the intestinal mucosal immune response in the
offspring by decreasing the CD8 IEL percentage.
Discussion
Retinol is the main form of ingested VA, and it has become the
diagnostic criteria for the serum in many clinical applications. In
the present study, as compared with a VAD model established
after weaning [26] or mid gestation [27], we successfully constructed a VAD animal model in the rat from the beginning of the
gestation period, which is consistent with Jiangs model [17], and
we obtained VAD offspring with approximately 0.3 mmol/L of
serum retinol after birth. There were no signicant differences in

the VAD and VAN groups with regard to time to delivery, number
of pups per litter, birth weight, and pup physiologic state;
however, a small body size with messy, dry, and dim fur was
observed in the VAD pups 6 wk postnatally (data not shown). To
conrm the effects of VAD during the gestational period, we
designed the VAD-N group in this study to be decient for VA
during gestation by cross-fostering VAD pups to VAN dams to
create the VAD-N pups. The increase in the serum retinol level in
the VAD-N pups further indicated that the postnatal feeding of
milk from VAN female rats could restore the lower retinol levels
caused by gestational VAD (Fig. 2A), thereby providing a useful
animal model that simulated VAD in pregnant women. Although
the RA levels were affected by both the VA (P < 0.0001) and LPS
effects (P 0.0077), VAD during the early life period decreased
the serum RA level in the offspring; this pattern was independent of LPS treatment (Fig. 2B).
IgA is synthesized by the plasma cells and epithelium of the
intestine, and it prevents pathogens and their toxic products
from attaching to the mucosal surface of the intestine [28]. The
level of IgA in the feces of VAD rats was markedly lower than that
of VAN rats [29]. The article by Quadro et al. [30] showed that the
IgA levels in the retinol-binding protein knockout mice were half
those of the wild-type mice. In the present study, there were no
differences among the VAN, VAD, and VAD-N groups in the
absence of LPS treatment. IgA is an important immunoglobulin in
the intestine that protects against pathogen invasion, and an
increase in the IgA level can be induced by exogenous pathogens.
After the injection of 3 mg/kg of LPS for 12 h, we found that the
rats displayed obvious congestion and edema of the small intestine, with yellow feces, diarrhea, and even twitch (data not

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X. Liu et al. / Nutrition 30 (2014) 350357

shown); there was also a prompt increase in the IgA levels of the
VAN pups after LPS exposure (Fig. 2C). These results demonstrated that the LPS abdominal challenge caused relevant clinical
features. The levels of IgA were considerably suppressed by the
LPS challenge in the both VAD and VAD-N pups as compared
with the VANLPS pups, which suggests that VAD during
gestation impairs a normal IgA response in the gut. VAD can
result in a decreased level of RA, which impairs the expression of
retinol-binding protein, the homing and maturation of B cells,
and the synthesis of immunoglobulin [15,30].
CD4CD25 T cells play a critical role in maintaining intestinal homeostasis and in suppressing systemic and mucosal immune activities in the gut. The percentage of CD4CD25 T cells
in the spleen was increased by the LPS challenge (Fig. 3B), which
suggests that LPS injection into the abdomen can upregulate the
number of CD4CD25 T cells and that the LPS main effect on the
number change of CD4CD25 T cells in the spleen was independent of VAD. The CD4CD8 DP T cells, which are antigenspecic effective cells, can activate memory cells, and they are
likely involved in the regulation of immune responses [31]. We
therefore hypothesized that the depression of CD4CD8 DP T
cells in both the VAD and VAD-N pups further caused dysfunction of the immune responses. These ndings demonstrated that
LPS-effectindependent VA treatment could induce an increase
in the number of CD4CD25 T cells and that both LPS challenge
and VAD during gestation may impair the normal systemic immune response through a decrease in the number of CD4CD8
DP T cells.
Unlike the decrease in T-cell subsets in the spleen, the number of B lymphocytes in the MLN was signicantly increased by
gestational VAD; this pattern was independent of LPS treatment
(Fig. 4). This is consistent with the ndings of Change et al. [32],
in which the number of immunoglobulin-Gsecreting B cells was
increased in the MLN of VAD mice. RA can upregulate the levels
of integrin a4b7 and the CCR9 expression of B lymphocytes [33],
which enhances the preferential homing of the B lymphocytes to
the small intestine and induces the secretion of IgA. We therefore
speculate that the decreased homing signaling by RA causes
fewer B cells to travel from the MLN to the gut.
The intestinal PPs are collective mucosal lymphoid tissues,
and they are the most important inductive sites during the
mucosal immune response. The distribution of lymphocyte
populations, which was identied with the use of ow cytometry
in the present study, was consistent with previous data [34,35].
DCs are the classic antigen-presenting cells, and immature DCs
are dispersed within the subepithelial domes of the PPs. Contrary to the report by Dong et al. [27], we found that the number
of CD11 C DCs in the PPs was reduced in the VAD pups (Fig. 5A);
this is due to the use of VAD rats from the beginning of gestation
and the different markers of CD11 C DCs that were used in the
present study. VA is one of the key factors during the differentiation of CD4CD25 T cells from naive T cells, and RA activates
the transforming growth factor-b signaling pathway to enhance
the maturation of Foxp3 regulatory T cells [36,37]. The data
obtained in the present study showed that the percentages of
both CD11 C DCs and CD4CD25 T cells were signicantly
suppressed by gestational VAD independent of the LPS challenge
(Fig. 5). These results suggest that VAD during the gestation
period limits the activities of inducible CD11 C DCs and
CD4CD25 T cells in the PPs.
IELs are effective T lymphocytes that are part of the mucosal
immune response of the intestine, and they are scattered in the
basal aspect of the intestinal epithelium. Most of the IELs are
CD8 T lymphocytes, and more than 85% of the isolated IELs in

this study were CD8 T cells (Fig. 6A), which is consistent with
previous data about lymphocyte populations [21]. The CD8 IELs
display characteristics that are similar to cytotoxic T lymphocytes, and IELs with TCRgd perform activities that are similar to
those of natural killer cells. As shown in Figure 6B, the percentage of CD8 IELs was signicantly decreased in the combined
VAD offspring, thus indicating that the immunologic activity of
the IELs was suppressed by the early life VAD. RA signaling
regulates the differentiation of T cells as well as the homing of T
and B cells in the intestine [3840]. Furthermore, IELs have been
implicated in antibody class switching and IgA production [25].
Fujihashi et al. [41] reported that the gut epithelium was
abnormal in TCRgd-decient mice, that the level of major histocompatibility complex class II molecule expression in the
enterocytes was reduced, and that the mucosal IgA production
was impaired. Therefore, the decrease in TCRgd IELs may
explain the repression of IgA secretion in the combined VAD-N
pups. The ndings from the present study suggest that the
mucosal immune functions of IELs are weakened by VAD during
the early life period. In the present study, the combined VAD-N
pups had a lower percentage of TCRgd IELs as compared with
the combined VAN pups; this decrease was comparable with or
without the LPS challenge.
We were surprised to nd that the results of the series of
parameters in the VAD-N group were the same as those of the
VAD group, including the decrease seen in IgA levels after LPS
treatment (Fig. 2C), the depression of splenic CD4CD8 DP T
cells without LPS challenge (Fig. 3C), the reduction in CD11 C
DCs (Fig. 5B) and CD4CD25 T cells (Fig. 5D) in the PP, and the
increase in B cells in the MLN (Fig. 4B). These results provide
novel evidence that the nutritional level of VA during the
gestational period plays a critical role in the immune responses.
Further studies involving this approach should focus on the
regulatory mechanisms of VA for the mucosal immunity of the
intestine and the identication of the best time point and dosage
for VA supplementation.
Conclusions
VAD during the gestational period may impair the intestinal
mucosal immune responses of offspring by suppressing the
numbers of CD11 C DCs and CD4CD25 T cells in the PPs and by
increasing the number of B cells in the MLNs. Early life VAD
impairs the number of CD8 IELs and decreases the serum retinol
level. IgA secretion and the percentage of splenic CD4CD8 T
cells were affected by both the VA and LPS treatments. These
ndings indicate that it is important to focus on the nutritional
states of both children and gestating women.
Acknowledgments
This work was supported by grants from National Nature
Science of Foundation of China (81070286) and Scientic
Research Foundation for the Returned Overseas Chinese Scholars,
State Education Ministry (2009-1001), as well as Science &
Technology Project of Yuzhong District of Chongqing (2012) to
Professor Jie Chen.
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