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 (2–4). 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 (5–7). 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.
Materials and Methods
Mice and immunizations
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.
Tissue preparation, flow cytometry, and microscopy/immunofluorescence
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).
ELISA and ELISPOT analyses
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).
Fluorescence in situ hybridization combined with flow cytometry
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).
Results and Discussion
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, 15–17).
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.
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.
The authors have no financial conflicts of interest.