Retinoic acid (RA) is a critical regulator of the intestinal adaptive immune response. However, the intrinsic impact of RA on B cell differentiation in the regulation of gut humoral immunity in vivo has never been directly shown. To address this issue, we have been able to generate a mouse model where B cells specifically express a dominant-negative receptor α for RA. In this study, we show that the silencing of RA signaling in B cells reduces the numbers of IgA+ Ab-secreting cells both in vitro and in vivo, suggesting that RA has a direct effect on IgA plasma cell differentiation. Moreover, the lack of RA signaling in B cells abrogates Ag-specific IgA responses after oral immunization and affects the microbiota composition. In conclusion, these results suggest that RA signaling in B cells through the RA receptor α is important to generate an effective gut humoral response and to maintain a normal microbiota composition.

All-trans retinoic acid (RA) is the main metabolite of vitamin A involved in immune regulation (1), where its primary function is to regulate gene transcription via binding to the nuclear RA receptors (RARs) and retinoic X receptors (1). It has been shown that vitamin A deficiency results in low IgA titers in the intestine, which is correlated with low IgA plasma cell numbers in the gut (24). Consistent with the effect of RA in IgA differentiation, it has been demonstrated that oral doses of RA agonists in rats increase IgA titers, inducible NO synthase expression, and nitrite/nitrate levels, which are important for IgA class switching (57). However, it is not known whether this is due to a direct effect on B cells or an indirect effect through follicular T cells and dendritic cells, which could support IgA plasma cell (PC) differentiation in the gut. We have used a mouse model in which RARα signaling is inhibited specifically in B cells by overexpressing a dominant-negative form of RARα. Using this model, we observed that the lack of RA signaling in B cells abrogated Ag-specific IgA responses after oral immunization and altered microbiota composition. In summary, these results definitively establish that RA signaling in B cells is critical in generating an effective gut humoral response and for the maintenance of a normal microbiota composition.

CD19Cre mice were purchased from The Jackson Laboratory. The dominant negative (dn)RARα mice have been previously described (8). Mice aged 8–10 wk were immunized with 10 μg of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP)–cholera toxin (CT) (provided by Nils Lycke) as described previously (9). These studies were approved and conducted in accredited facilities in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 (Home Office license no. PPL 70/7102). All animals were cohoused and maintained in a specific pathogen-free facility at King’s College London.

Tissues were processed and single-cell suspensions were prepared and stained with Abs for flow cytometry as well as for microscopy as described previously (10, 11). B cells were cultured in vitro as reported before (4). For the analysis of IgA binding bacteria, preparation of the stool, staining, and acquisition were done as described previously (12).

Total RNA extraction, real-time quantification, and analysis were performed as previously described (11). A TaqMan gene expression assay for mouse Aicda (Mm00507774_m1) was multiplexed with GAPDH endogenous control probe (4352339E) (Applied Biosystems).

Gut lavages and sera were used to assess NP-IgA, IgA, and IgG1 titers by ELISA according to the manufacturer’s protocol (mouse IgG1/IgA total ELISA Ready-SET-Go!, eBioscience). Single-cell preparations were obtained from mesenteric lymph node (MLN), Peyer’s patches (PP), spleen, and small intestinal lamina propria (sLP), and the number of NP-IgA or total IgA–Ab-secreting cells (ASCs) was determined by ELISPOT as reported previously (9, 11).

Stool samples were collected and fluorescence in situ hybridization combined with flow cytometry was performed as previously described (13). The EUB 338 probe (FITC labeled) was used as the positive control probe, the NON 338 probe (Cy5 labeled) was used as the negative control, and specific probes (Cy5 labeled) were used for identification of subgroups (14).

To analyze the functional impact of intrinsic RA signaling during B cell differentiation, a dominant-negative form of RARα was overexpressed in B cells by interbreeding dnRARαfl/fl (8) mice with CD19Cre mice (hereafter denoted dnRARαCD19Cre). Analysis by quantitative PCR showed that only B cells from dnRARαCD19Cre mice expressed the dnRARα form (Fig. 1A). To test that our model was able to inhibit RA signaling in B cells, we performed in vitro culture experiments, as previously described (4). Briefly, splenic B cells from dnRARαCD19Cre or dnRARα mice were enriched and activated in vitro with anti-mouse IgM in the presence or absence of 10 nM RA. Our results showed that when RA signaling was abrogated in B cells, these cells were not able to induce α4β7 expression (Fig. 1B, 1C) or generate IgA-ASCs (Fig. 1B, 1D) compared with control cells. These data demonstrate that RA signaling is necessary to induce gut homing receptors in B cells and IgA-PC differentiation in vitro, which is consistent with several independent reports (4, 1517).

FIGURE 1.

RA signaling in B cells is essential to induce α4β7 expression and IgA PCs. (A) dnRARα gene expression from purified splenic B cells, T cells, and monocytes from dnRARαCD19Cre mice and littermate controls. (B) Representative dot plots of α4β7+ B cells and IgA+ PCs from splenic B cells from dnRARαCD19Cre and littermate controls after 5 d of culture with anti-mouse IgM in the presence or absence of 10 nM RA. For the analysis of α4β7, the cells were gated in live B220+ B cells (and CD11c to discard dendritic cells), and for the detection of IgA+ ASCs the gates were live B220CD138+ cells. (C) Quantification of α4β7 expression in live B220+ B cells. (D) Percentage of IgA+ PCs in live B220CD138+ cells. (EG) Analysis of B cells from PP of dnRARαCD19Cre mice was performed. Percentage of B220+ B cells (E), percentage of GC B cells (F), and IgA+ GC B cells (G) are shown. Cells were analyzed by flow cytometry and gated in live B220+ B cells. For the detection of IgA+ GC B cells, cells were gated as live B220+CD95+GL-7+. Littermates were used as a control. (H) Aicda gene expression was analyzed in GC B cells from PP of dnRARαCD19Cre and control mice by quantitative PCR. Bar graph represent mean ± SEM. ***p < 0.001; n = 9–10 mice/group.

FIGURE 1.

RA signaling in B cells is essential to induce α4β7 expression and IgA PCs. (A) dnRARα gene expression from purified splenic B cells, T cells, and monocytes from dnRARαCD19Cre mice and littermate controls. (B) Representative dot plots of α4β7+ B cells and IgA+ PCs from splenic B cells from dnRARαCD19Cre and littermate controls after 5 d of culture with anti-mouse IgM in the presence or absence of 10 nM RA. For the analysis of α4β7, the cells were gated in live B220+ B cells (and CD11c to discard dendritic cells), and for the detection of IgA+ ASCs the gates were live B220CD138+ cells. (C) Quantification of α4β7 expression in live B220+ B cells. (D) Percentage of IgA+ PCs in live B220CD138+ cells. (EG) Analysis of B cells from PP of dnRARαCD19Cre mice was performed. Percentage of B220+ B cells (E), percentage of GC B cells (F), and IgA+ GC B cells (G) are shown. Cells were analyzed by flow cytometry and gated in live B220+ B cells. For the detection of IgA+ GC B cells, cells were gated as live B220+CD95+GL-7+. Littermates were used as a control. (H) Aicda gene expression was analyzed in GC B cells from PP of dnRARαCD19Cre and control mice by quantitative PCR. Bar graph represent mean ± SEM. ***p < 0.001; n = 9–10 mice/group.

Close modal

It has been previously demonstrated that vitamin A deficiency affects generation of IgA-ASCs in the gut; however, it is unknown whether RA has a direct effect on B cells in vivo. Therefore, we evaluated the development of germinal center (GC) B cells in PP of dnRARαCD19Cre mice. To exclude variation due to housing conditions, in all in vivo experiments, dnRARαCD19Cre mice and littermate controls were cohoused. We observed that the number of PP and the percentage as well as the absolute number of B220+ cells in PP from dnRARαCD19Cre mice were normal compared with control mice (Fig. 1E and data not shown). We also observed an increase in the percentage and absolute number of CD95+GL-7+ GC B cells in the PP from dnRARαCD19Cre mice (Fig. 1F and data not shown). Surprisingly, we found a reduction in IgA+ GC B cells as a percentage and absolute number in PP of dnRARαCD19Cre mice (Fig. 1G and data not shown). Additionally, Aicda gene expression analyzed by quantitative PCR was lower in GC B cells from PP of dnRARαCD19Cre when compared with that from control mice (Fig. 1H), suggesting that the IgA+ GC B cell reduction observed was due to a reduction in isotype switching. Taken together, these data indicate that RA signaling in B cells is necessary to maximize the number and frequency of IgA+ GC B cells.

Because RA signaling in B cells is important for the induction of IgA+ GC B cells in PP, we analyzed its role in the generation of IgA-ASCs. We found a drastic reduction in IgA-ASC number in the small intestine of dnRARαCD19Cre mice compared with control mice (Fig. 2A, 2B). This reduction was correlated with low IgA titers found in intestinal secretions from dnRARαCD19Cre mice (Fig. 2C). Furthermore, we also observed a reduction in the percentage of IgA bound to bacteria (Fig. 2D). We then explored the immune response to the well-characterized hapten NP-CT (9). Following NP-CT oral immunization, specific IgA responses in the small intestine of dnRARαCD19Cre and control mice were analyzed. Frequency of NP-specific ASCs in the sLP, PP, and MLN was reduced when RA signaling was abrogated in B cells (Fig. 2E). We also observed a reduction in NP-IgA titers found in the stool of dnRARαCD19Cre compared with control mice (Fig. 2F), whereas systemic NP-IgG1 titer and frequency of NP-specific IgA-ASCs in the spleen remained unaltered (Fig. 2E, 2G). Additionally, we did not observe an accumulation of NP-IgM ASCs in the gut (data not shown). Taken together, these results indicate that RA signaling in B cells plays an important role in generating Ag-specific IgA responses in the gut.

FIGURE 2.

RA signaling in B cells is required for effective IgA-PC differentiation and normal microflora maintenance in the gut. (A) Histology showing IgA+ cells in sLP from dnRARαCD19Cre and control mice (n = 4/group). (B) Single-cell suspensions were obtained from sLP of dnRARαCD19Cre mice and littermate controls and the number of IgA-ASCs was determined by ELISPOT. (C) Quantification of luminal IgA levels from sLP of dnRARαCD19Cre mice measured by ELISA (n = 8/group). (D) Percentage of IgA-binding bacteria from small intestine of dnRARαCD19Cre and littermate controls. (E) Mice were orally immunized twice with NP-CT or saline solution alone. Cells were then isolated from MLN, PP, spleen, and sLP and the number of NP-IgA+ PCs was determined by ELISPOT. (F) Quantification of NP-IgA titers in the stool from sLP of dnRARαCD19Cre mice and controls measured by ELISA. (G) Quantification of NP-IgG1 titers in serum by ELISA. (H) Fecal samples were collected from dnRARαCD19Cre and control mice and the different bacterial groups (BAC, Bacteroides and Prevotella; BIF, Bifidobacterium; EREC, Lachnospiraceae; LAB, Lactobacillus and Streptococcus) were identified by fluorescence in situ hybridization–flow cytometry (n = 14/group). Bar graph represents mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

RA signaling in B cells is required for effective IgA-PC differentiation and normal microflora maintenance in the gut. (A) Histology showing IgA+ cells in sLP from dnRARαCD19Cre and control mice (n = 4/group). (B) Single-cell suspensions were obtained from sLP of dnRARαCD19Cre mice and littermate controls and the number of IgA-ASCs was determined by ELISPOT. (C) Quantification of luminal IgA levels from sLP of dnRARαCD19Cre mice measured by ELISA (n = 8/group). (D) Percentage of IgA-binding bacteria from small intestine of dnRARαCD19Cre and littermate controls. (E) Mice were orally immunized twice with NP-CT or saline solution alone. Cells were then isolated from MLN, PP, spleen, and sLP and the number of NP-IgA+ PCs was determined by ELISPOT. (F) Quantification of NP-IgA titers in the stool from sLP of dnRARαCD19Cre mice and controls measured by ELISA. (G) Quantification of NP-IgG1 titers in serum by ELISA. (H) Fecal samples were collected from dnRARαCD19Cre and control mice and the different bacterial groups (BAC, Bacteroides and Prevotella; BIF, Bifidobacterium; EREC, Lachnospiraceae; LAB, Lactobacillus and Streptococcus) were identified by fluorescence in situ hybridization–flow cytometry (n = 14/group). Bar graph represents mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

The importance of crosstalk between the microbiome and the immune system in IgA-mediated intestinal homeostasis has been previously described (18). Because dnRARαCD19Cre and control mice have different humoral IgA responses, we investigated whether the gut microflora was altered in these mice. To address this, fecal samples from cohoused dnRARαCD19Cre and dnRARα mice were analyzed. Composition of gut microbiota was significantly different between dnRARαCD19Cre and control mice (Fig. 2H). The dnRARαCD19Cre mice displayed an increase in the proportion of adherent bacteria Lachnospiraceae (Erec482+) and Lactobacillus/Streptococcus (Lab158+) groups compared with control mice, which has been suggested to be associated with colorectal adenomas (19). Taken together, our results demonstrate that altered IgA responses, due to lack of RA signaling in B cells, significantly affects the symbiotic relationship between host and commensal bacteria in the gut.

In conclusion, to our knowledge, this is the first time that a direct effect of RA signaling in B cells has been shown in vivo. Our results demonstrate that RA signaling in B cells is not essential for their homing to PP. However, it is necessary to maintain an optimal IgA humoral immune response and a normal microbiota composition in the gut. Overall, these results further support the potential use of RA as an adjuvant in preventing dietary allergies as previously suggested by others (20, 21).

This work was supported by Wellcome Trust Grant WT091823/z/10/z (to R.J.N.). The work was also supported by the National Institute for Health Research Biomedical Research Centre at Guy’s and St. Thomas’ National Health Service Foundation Trust and King’s College London. The views expressed are those of the authors and not necessarily those of the National Health Service, the National Institute for Health Research, or the Department of Health.

Abbreviations used in this article:

     
  • ASC

    Ab-secreting cell

  •  
  • CT

    cholera toxin

  •  
  • dn

    dominant-negative

  •  
  • GC

    germinal center

  •  
  • MLN

    mesenteric lymph node

  •  
  • NP

    (4-hydroxy-3-nitrophenyl)acetyl

  •  
  • PC

    plasma cell

  •  
  • PP

    Peyer’s patch

  •  
  • RA

    retinoic acid

  •  
  • RAR

    RA receptor

  •  
  • sLP

    small intestinal lamina propria.

1
Mora
J. R.
,
Iwata
M.
,
von Andrian
U. H.
.
2008
.
Vitamin effects on the immune system: vitamins A and D take centre stage.
Nat. Rev. Immunol.
8
:
685
698
.
2
McDermott
M. R.
,
Mark
D. A.
,
Befus
A. D.
,
Baliga
B. S.
,
Suskind
R. M.
,
Bienenstock
J.
.
1982
.
Impaired intestinal localization of mesenteric lymphoblasts associated with vitamin A deficiency and protein-calorie malnutrition.
Immunology
45
:
1
5
.
3
Bjersing
J. L.
,
Telemo
E.
,
Dahlgren
U.
,
Hanson
L. A.
.
2002
.
Loss of ileal IgA+ plasma cells and of CD4+ lymphocytes in ileal Peyer’s patches of vitamin A deficient rats.
Clin. Exp. Immunol.
130
:
404
408
.
4
Mora
J. R.
,
Iwata
M.
,
Eksteen
B.
,
Song
S.-Y.
,
Junt
T.
,
Senman
B.
,
Otipoby
K. L.
,
Yokota
A.
,
Takeuchi
H.
,
Ricciardi-Castagnoli
P.
, et al
.
2006
.
Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells.
Science
314
:
1157
1160
.
5
Kuwabara
K.
,
Shudo
K.
,
Hori
Y.
.
1996
.
Novel synthetic retinoic acid inhibits rat collagen arthritis and differentially affects serum immunoglobulin subclass levels.
FEBS Lett.
378
:
153
156
.
6
Seguin-Devaux
C.
,
Devaux
Y.
,
Latger-Cannard
V.
,
Grosjean
S.
,
Rochette-Egly
C.
,
Zannad
F.
,
Meistelman
C.
,
Mertes
P.-M.
,
Longrois
D.
.
2002
.
Enhancement of the inducible NO synthase activation by retinoic acid is mimicked by RARα agonist in vivo.
Am. J. Physiol. Endocrinol. Metab.
283
:
E525
E535
.
7
Devaux
Y.
,
Grosjean
S.
,
Seguin
C.
,
David
C.
,
Dousset
B.
,
Zannad
F.
,
Meistelman
C.
,
De Talancé
N.
,
Mertes
P. M.
,
Ungureanu-Longrois
D.
.
2000
.
Retinoic acid and host-pathogen interactions: effects on inducible nitric oxide synthase in vivo.
Am. J. Physiol. Endocrinol. Metab.
279
:
E1045
E1053
.
8
Rajaii
F.
,
Bitzer
Z. T.
,
Xu
Q.
,
Sockanathan
S.
.
2008
.
Expression of the dominant negative retinoid receptor, RAR403, alters telencephalic progenitor proliferation, survival, and cell fate specification.
Dev. Biol.
316
:
371
382
.
9
Bergqvist
P.
,
Stensson
A.
,
Hazanov
L.
,
Holmberg
A.
,
Mattsson
J.
,
Mehr
R.
,
Bemark
M.
,
Lycke
N. Y.
.
2013
.
Re-utilization of germinal centers in multiple Peyer’s patches results in highly synchronized, oligoclonal, and affinity-matured gut IgA responses.
Mucosal Immunol.
6
:
122
135
.
10
Elgueta
R.
,
Sepulveda
F. E.
,
Vilches
F.
,
Vargas
L.
,
Mora
J. R.
,
Bono
M. R.
,
Rosemblatt
M.
.
2008
.
Imprinting of CCR9 on CD4 T cells requires IL-4 signaling on mesenteric lymph node dendritic cells.
J. Immunol.
180
:
6501
6507
.
11
Elgueta
R.
,
Marks
E.
,
Nowak
E.
,
Menezes
S.
,
Benson
M.
,
Raman
V. S.
,
Ortiz
C.
,
O’Connell
S.
,
Hess
H.
,
Lord
G. M.
,
Noelle
R.
.
2015
.
CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen.
J. Immunol.
194
:
505
513
.
12
Kawamoto
S.
,
Tran
T. H.
,
Maruya
M.
,
Suzuki
K.
,
Doi
Y.
,
Tsutsui
Y.
,
Kato
L. M.
,
Fagarasan
S.
.
2012
.
The inhibitory receptor PD-1 regulates IgA selection and bacterial composition in the gut.
Science
336
:
485
489
.
13
Rigottier-Gois
L.
,
Bourhis
A. G.
,
Gramet
G.
,
Rochet
V.
,
Doré
J.
.
2003
.
Fluorescent hybridisation combined with flow cytometry and hybridisation of total RNA to analyse the composition of microbial communities in human faeces using 16S rRNA probes.
FEMS Microbiol. Ecol.
43
:
237
245
.
14
Powell
N.
,
Walker
A. W.
,
Stolarczyk
E.
,
Canavan
J. B.
,
Gökmen
M. R.
,
Marks
E.
,
Jackson
I.
,
Hashim
A.
,
Curtis
M. A.
,
Jenner
R. G.
, et al
.
2012
.
The transcription factor T-bet regulates intestinal inflammation mediated by interleukin-7 receptor+ innate lymphoid cells.
Immunity
37
:
674
684
.
15
Tsuji
M.
,
Suzuki
K.
,
Kitamura
H.
,
Maruya
M.
,
Kinoshita
K.
,
Ivanov
I. I.
,
Itoh
K.
,
Littman
D. R.
,
Fagarasan
S.
.
2008
.
Requirement for lymphoid tissue-inducer cells in isolated follicle formation and T cell-independent immunoglobulin A generation in the gut.
Immunity
29
:
261
271
.
16
Suzuki
K.
,
Maruya
M.
,
Kawamoto
S.
,
Sitnik
K.
,
Kitamura
H.
,
Agace
W. W.
,
Fagarasan
S.
.
2010
.
The sensing of environmental stimuli by follicular dendritic cells promotes immunoglobulin A generation in the gut.
Immunity
33
:
71
83
.
17
Watanabe
K.
,
Sugai
M.
,
Nambu
Y.
,
Osato
M.
,
Hayashi
T.
,
Kawaguchi
M.
,
Komori
T.
,
Ito
Y.
,
Shimizu
A.
.
2010
.
Requirement for Runx proteins in IgA class switching acting downstream of TGF-β1 and retinoic acid signaling.
J. Immunol.
184
:
2785
2792
.
18
Shulzhenko
N.
,
Morgun
A.
,
Hsiao
W.
,
Battle
M.
,
Yao
M.
,
Gavrilova
O.
,
Orandle
M.
,
Mayer
L.
,
Macpherson
A. J.
,
McCoy
K. D.
, et al
.
2011
.
Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut.
Nat. Med.
17
:
1585
1593
.
19
Shen
X. J.
,
Rawls
J. F.
,
Randall
T.
,
Burcal
L.
,
Mpande
C. N.
,
Jenkins
N.
,
Jovov
B.
,
Abdo
Z.
,
Sandler
R. S.
,
Keku
T. O.
.
2010
.
Molecular characterization of mucosal adherent bacteria and associations with colorectal adenomas.
Gut Microbes
1
:
138
147
.
20
Indrevær
R. L.
,
Holm
K. L.
,
Aukrust
P.
,
Osnes
L. T.
,
Naderi
E. H.
,
Fevang
B.
,
Blomhoff
H. K.
.
2013
.
Retinoic acid improves defective TLR9/RP105-induced immune responses in common variable immunodeficiency-derived B cells.
J. Immunol.
191
:
3624
3633
.
21
DePaolo
R. W.
,
Abadie
V.
,
Tang
F.
,
Fehlner-Peach
H.
,
Hall
J. A.
,
Wang
W.
,
Marietta
E. V.
,
Kasarda
D. D.
,
Waldmann
T. A.
,
Murray
J. A.
, et al
.
2011
.
Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens.
Nature
471
:
220
224
.

The authors have no financial conflicts of interest.