Diet-Derived Short Chain Fatty Acids Stimulate Intestinal Epithelial Cells to Induce Mucosal Tolerogenic Dendritic Cells

Diet-Derived Short Chain Fatty Acids Stimulate Intestinal Epithelial Cells To Induce Mucosal Tolerogenic Dendritic Cells

This information is current as Gera Goverse, Rosalie Molenaar, Laurence Macia, Jian Tan, of September 27, 2021. Martje N. Erkelens, Tanja Konijn, Marlene Knippenberg, Emma C. L. Cook, Diana Hanekamp, Marc Veldhoen, Anita Hartog, Guus Roeselers, Charles R. Mackay and Reina E. Mebius

J Immunol 2017; 198:2172-2181; Prepublished online 18 Downloaded from January 2017; doi: 10.4049/jimmunol.1600165 http://www.jimmunol.org/content/198/5/2172 http://www.jimmunol.org/ References This article cites 53 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/198/5/2172.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Diet-Derived Short Chain Fatty Acids Stimulate Intestinal Epithelial Cells To Induce Mucosal Tolerogenic Dendritic Cells

Gera Goverse,* Rosalie Molenaar,* Laurence Macia,† Jian Tan,† Martje N. Erkelens,* Tanja Konijn,* Marlene Knippenberg,* Emma C. L. Cook,* Diana Hanekamp,* Marc Veldhoen,‡ Anita Hartog,x,{ Guus Roeselers,‖ Charles R. Mackay,† and Reina E. Mebius*

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article, we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced Downloaded from the expression of the vitamin A–converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro, respectively. Furthermore, our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A–converting enzymes in mesenteric lymph node dendritic cells, along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover, we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion, our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system. The Journal of http://www.jimmunol.org/ Immunology, 2017, 198: 2172–2181.

he immune system is influenced by its immediate sur- within the lumen are fundamental to facilitate this process and to roundings, because nutritional intake and dietary changes allow the uptake of all of the necessary nutrients from the diet. They T have large effects on the intestinal and systemic immune are able to process diet-derived products in metabolites that may system. Studies showed that diet-derived products can influence the otherwise not be digestible by the host (10, 11). The highest immune system within the lamina propria of the intestine in a bacterial load in the gastrointestinal tract is found within the co- beneficial manner (1–4). However, dietary intake can also lead to lon. Although the number of bacteria is much lower in the small immune-mediated disorders, such as type 2 diabetes and inflam- intestines (SIs), it was shown that many species of bacteria are by guest on September 27, 2021 matory bowel disease (1, 4–7). Therefore, the diet in Western present in the SIs (12–14). In addition, the distribution of microbiota countries may underlie immunological disorders, such as food within the intestines can be influenced by the available nutrients in allergies, asthma, and certain autoimmune diseases (8, 9). the diet. Many studies, using high-throughput sequencing of gut The intestines provide the body with essential nutrients by di- microbiota, identified specific bacterial taxa that are beneficial for the gestion and absorption of dietary products. Microbiota located host or might be associated with immune disorders (7, 13, 15, 16). Various anaerobic bacteria produce short chain fatty acids *Department of Molecular Cell Biology and Immunology, VU University Medical (SCFAs), predominantly acetate, butyrate, and propionate, upon Center, 1081 HZ Amsterdam, the Netherlands; †Department of Immunology, Monash metabolism of dietary fiber. The production of these SCFAs is University, Clayton, Victoria 3800, Australia; ‡Babraham Institute, Cambridge CB22 x { considered a benefit to the host. Although SCFAs are found at 3AT, United Kingdom; Nutricia Research, 3584 CT Utrecht, the Netherlands; Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CG Utrecht, the higher concentrations in the colon, they are also present in the ‖ Netherlands; and Microbiology and Systems Biology, TNO, 3704 HE Zeist, the cecum and SI (12, 14). SCFAs, in particular butyrate, form the Netherlands major energy source for intestinal epithelial cells (IECs). In ad- ORCIDs: 0000-0002-9467-8693 (M.N.E.); 0000-0001-9834-7202 (M.K.); 0000- dition, SCFAs are described to promote the barrier function of 0002-1415-4049 (E.C.L.C.); 0000-0002-5902-1127 (D.H.); 0000-0002-1478- 9562 (M.V.); 0000-0002-4725-6105 (G.R.); 0000-0002-6338-7340 (C.R.M.). IECs (10, 17). Recently, multiple studies showed the beneficial Received for publication January 27, 2016. Accepted for publication December 16, effect of SCFAs on intestinal regulatory T cells and during in- 2016. testinal inflammation (18, 19); however, the exact mechanisms of This work was supported by the Dutch Scientific Research program (Netherlands this regulation are not completely understood. Organisation for Scientific Research Grant VICI [918.56.612] and ALW-TOP Grant It is necessary that immunological tolerance toward harmless [854.10.005] to R.E.M.) and by the National Health and Medical Research Council of Australia (Grant 1068890). food Ags and commensal flora is established in the intestines, Address correspondence and reprint requests to Prof. Reina E. Mebius, VU Univer- whereas immunity against pathogens is crucial for host protection. sity Medical Center, OI2 Building, Boelelaan 1108, 1081HZ Amsterdam, the Nether- Tolerogenic dendritic cells (DCs) are well-described immune cells lands. E-mail address: [email protected] that interact with other hematopoietic cells to maintain a balanced Abbreviations used in this article: Ahr, aryl hydrocarbon receptor; ALDH, aldehyde mucosal immune system (20, 21). These tolerogenic DCs express dehydrogenase; 4-CMTB, 4-chloro-a-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide; DC, dendritic cell; DIM, 3,39-diindolymethane; HDAC, histone deacetylase; IEC, intestinal CD103 and are able to sample Ags in the intestinal lamina propria epithelial cell; MLN, mesenteric lymph node; qPCR, quantitative PCR; RA, retinoic acid; and migrate to the draining mesenteric lymph node (MLN) to RALDH, retinaldehyde dehydrogenase; SCFA, short chain fatty acid; SI, small intestine; present Ag and to activate lymphocytes. With the production of TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TSA, trichostatin A; WT, wild-type. retinoic acid (RA), the active metabolite of vitamin A, these DCs Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 are crucial for the differentiation of regulatory T cells and promote www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600165 The Journal of Immunology 2173

IgA class-switching of B cells and induce the expression of gut- 59-CTGCGGACTGTCCCATCAATG-39, reverse: 59-GCAGCTCACCAA- homing molecules on lymphocytes (20–22). We (23) and other CCACAGTTTCT-39) and primers for the housekeeping genes Ubiquitin C 9 9 9 investigators (24–26) showed that these tolerogenic DCs them- (forward: 5 -AGCCCAGTGTTACCACCAAG-3 , reverse: 5 -ACCCAA- GAACAAGCACAAGG-39) and Cyclo (forward: 59-ACCCATCAAAC- selves depend on RA, which is produced by IECs, for their CATTCCTTCTGTA-39,reverse:59-TGAGGAAAATATGGAACCCAAAGA-39) function and phenotype. The critical role of retinol metabolism by (Invitrogen) were used. Quantitative PCR (qPCR) analysis was performed small intestinal epithelium was shown by the absence of tolero- as described earlier (28). genic DCs in CRBPII-deficient mice, because CRBP is predom- Preparation of SI cell and lymph node suspensions inantly expressed in small IECs and is necessary for the uptake of retinol (26). SIs were dissected and opened longitudinally after removal of Peyer’s In this article, we report that fiber-enriched diets promote vitamin patches. SIs were treated with EDTA for the removal of epithelial cells and A metabolism in epithelial cells in the SIs, as well as enhanced digested to obtain a single-cell suspension, as previously described (21, 28). Subsequently, cell suspensions were immunomagnetically purified for tolerogenic activity in MLN DCs, increased percentages of in- CD45+ cells with PE-Cy7–labeled anti-CD45 (clone 30-F11; eBioscience/ testinal regulatory T cells, and higher levels of IgA production in Immunosource, Halle-Zoersel, Belgium) and the EasySep PE Positive the lumen of the intestine. Furthermore, we demonstrate that Selection Kit (STEMCELL Technologies, Grenoble, France). Single-cell SCFAs and the inhibition of histone deacetylase (HDAC) activity suspensions were made by cutting MLNs with scissors, followed by digestion at 37˚C for 25 min with constant stirring, using 0.5 mg/ml are able to induce this vitamin A metabolism in a small IEC line. Blendzyme 2 and 0.2 mg/ml DNase I (both from Roche, Penzberg, Ger- Strikingly, the immune system is altered upon dietary adjustments, many) in PBS. and the composition of the microbiota and SCFA levels within the

SI are also affected. Together, these ﬁndings reveal a delicate Flow cytometry and ALDEFLUOR assay Downloaded from mechanism that safeguards a tolerant mucosal immune system as a Aldehyde dehydrogenase (ALDH) activity in MLN cells was measured result of dietary intake. using an ALDEFLUOR Kit (STEMCELL Technologies), according to the manufacturer’s protocol. For ﬂow cytometric analysis of ALDEFLUOR- reacted cells, cells were subsequently stained with anti-CD45–PE–Cy7 Materials and Methods (clone 30F11), anti-CD11c–PE (clone N418; both from eBioscience), anti- Mice CD103–biotin (clone M290; BD Biosciences, Breda, the Netherlands),

MHC class II (clone M5/114, affinity-purified from hybridoma cell culture http://www.jimmunol.org/ Specific pathogen–free C57BL/6 mice (Charles River, Maastricht, the supernatants), Percp-conjugated (BD Biosciences) streptavidin, and Netherlands) were raised on a standard conventional 2016 Teklad Global SYTOX Blue (Invitrogen) to discriminate between live and dead cells. In rodent diet (Harlan Laboratories) or a synthetic diet that was based on addition, small intestinal cells were stained after CD45 selection with anti- AIN-93M with the use of vitamin-free casein (MP Biomedicals, Solon, CD3ε–488 (clone 145-2C11), anti-Foxp3–647 (clone FJK-16s), CD4– OH). Mice were kept under standard animal housing conditions. Wild-type Percp–Cy5.5 (clone RM4-5; all eBioscience), and LIVE/DEAD Fixable (WT) C57BL/6 mice, aged 8–14 wk, were sacrificed for collection of SIs. Near-IR (Invitrogen). Staining was performed using a Foxp3 staining set The mice fed a conventional diet were raised on a standard Teklad 2016 (eBioscience). Cells were analyzed with a Cyan ADP flow cytometer diet that contains 3.3% of crude fiber and is complemented with a stan- (Beckman Coulter, Mijdrecht, the Netherlands). dardized mineral and vitamin mix. Mice fed a synthetic diet received AIN- 93M, which contains 4000 IU/kg vitamin A. In another experiment, mice ELISA for secretory IgA were fed an AIN-93M diet containing 20,000 IU/kg of vitamin A. The by guest on September 27, 2021 Animal Experiments Committee of the VU Medical Center approved all Content from the SIs of mice was collected in cold PBS buffer. Debris was experiments described in this study. removed by cold centrifugation for 20 min at 2000 rpm to harvest the All diet studies were performed at Monash University. Mice were raised supernatant for analysis of secretory IgA, as described previously (28). on a synthetic AIN-93G diet that contains 3.2% fiber. At the age of 8–10 wk, mice were switched to a diet containing no fiber (0%, SF11-028; Special SCFA and lactic acid measurement Feeds) or high levels of fiber (35% enriched for guar gum and cellulose, SCFA and lactic acid measurements were performed on the same fecal SF11-029) for 2 wk. Sodium butyrate (Sigma-Aldrich) was administered in supernatants collected for IgA analysis, as described above. Acetic, pro- the drinking water at 100 mM for 3 wk before mice were sacrificed. All 2 2 pionic, and butyric acids were quantitatively determined using a Shimadzu- mice were backcrossed onto the C57BL/6 background; GPR43 / mice 2 2 GC2010 (Shimadzu) equipped with a flame ionization detector. The sample were obtained from Deltagen (San Mateo, CA), and GPR109 / mice (1 ml, split injection of 203) was injected at 90˚C into the column (ZB- were obtained from Offermans (Heidelberg, Germany). All experimental FFAB 15 m 3 0.53 3 1.0 mm; Phenomenex) using H as carrier gas. After procedures were carried out according to protocols approved by the rele- 2 injection of the sample, the oven was heated to 140˚C at a rate of 10˚C/ vant Animal Ethics Committees. min, followed by heating to 220˚C at 20˚C/min, and finally maintained at Cell culture temperature of 220˚C for 1 min. The temperature of the injector was 200˚C, and the detector was 250˚C. Lactate was determined enzymatically using The small IEC line mICcl2 was used for in vitro stimulation. Cells were a D-lactic acid/L-lactic acid test kit (Boehringer Mannheim/R-Biopharm, grown in a defined medium that was described earlier (27). Stimulations Darmstadt, Germany). were performed with sodium butyrate, sodium acetate, sodium propionate (all in a concentration of 1 mM after careful titration) and the HDAC Sequencing of 16S rRNA gene amplicons inhibitors trichostatin A (TSA; 300 nm), MS275 (2500 nm), M344 (5 mM), m and Droxinostat (50 mM) (all from Sigma-Aldrich). A 30-min pre- Small intestinal contents were added to 250 l of lysis buffer (Agowa, m m incubation with pertussis toxin (100 ng/ml; Sigma-Aldrich) was performed Berlin, Germany), 250 l zirconium beads (0.1 mm), and 200 l phenol, for the inhibition of GPCR signaling. 4-Chloro-a-(1-methylethyl)-N-2- followed by cell disruption by bead beating for 2 min. Extracted DNA was thiazolyl-benzeneacetamide (4-CMTB) (1 mm; Sigma-Aldrich) was used amplified and sequenced as described previously (29). as a GPR43 agonist. Other dietary- or flora-derived components that were All preprocessing and taxonomic classifications were performed using tested and titrated (see Fig. 3 for concentrations) included oleic acid, modules implemented in the mothur software platform (30), as performed conjugated linoleic acid (Lipid Nutrition, Wormerveer, the Netherlands), previously (31). Unique sequences were aligned using the align.seqs 25-hydroxycholesterol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,39- command and the mothur-compatible Bacterial SILVA SEED database. diindolymethane (DIM), and zymosan (all from Sigma-Aldrich). Sequences were classified taxonomically by the RDP-II Naive Bayesian Classifier using a 60% confidence threshold. Community profiles were RNA/DNA isolation and quantitative PCR compared by Weighted Unifrac clustering of OTU abundances (32). SIs were dissected, flushed with PBS, and homogenized in TRIzol Re- Statistics agent (Invitrogen, Breda, the Netherlands). RNA from mICcl2 cells was isolated and cDNA was synthesized as described previously (28). Spe- Results are given as the mean 6 SD. Statistical analyses were performed cific primers for aldh1a1 (forward: 59-CTCCTCTCACGGCTCTTCA-39, using the two-tailed Student t test or for multiple comparison or using one- reverse: 59-AATGTTTACCACGCCAGGAG-39)andnlpr3 (forward: way or two-way ANOVA for one or two variables, respectively. 2174 DIETARY FIBERS MODULATE THE MUCOSAL IMMUNE SYSTEM

Results specifically have a high expression of the RALDH1 enzyme, which Dietary intake affects mucosal immune system is needed for the production of RA to induce RALDH2 expression in DCs (21, 23, 24, 26, 33). To address whether RALDH1 ex- Vitamin A is synthesized into its active metabolite RA in two pression in IECs is influenced by external factors, we measured oxidative steps; the second step is irreversible and performed by aldh1a1 expression in the intestines of mice that were raised on a one of the three isoforms of retinaldehyde dehydrogenase (RALDH) standard conventional diet or a synthetic AIN-93M diet. Although enzymes: RALDH1–3. When we checked for the expression of the precise composition of the synthetic diet is known, the standard RALDH enzymes in small intestinal scrapings, which mainly conventional diet is composed of natural products that may contain consist of epithelial cells, we observed a high mRNA expression of unidentified components. However, the conventional diet is sup- aldh1a1 (Fig. 1A), the gene coding for RALDH1. These results are plemented with the same vitamin and mineral supplements com- in line with previously published data demonstrating that IECs parable to the levels found in the synthetic diet. SIs were collected Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Vitamin A metabolism in small IECs cells is affected by dietary intake. (A) aldh1a1 mRNA expression measured in total SI and epithelial scrapings from the SI. C57BL/6 mice were raised on a standard conventional diet or a synthetic AIN-93M diet. (B) The expression levels of aldh1a1 mRNA in proximal, middle, and distal parts of the SI. The levels were normalized to Ubiquitin and Cyclo mRNA levels. Relative mRNA expression levels of proximal SIs of mice fed a conventional diet were set at 1.0. ALDH activity was measured by flow cytometry in MLN DCs gated first for CD11c+CD45+ cells, followed by CD103+ expression on the forward scatter (FS) (C) and in MLN CD1032CD11c+ DCs (E) using the ALDEFLUOR assay in the absence or presence of ALDH inhibitor DEAB. (D) Percentages of CD103+ and CD1032 CD11c+ DCs in the MLN. (F) Bar graphs show ALDH activity measured in MLN CD103+ DCs of mice that received 4,000 or 20,000 IU/kg vitamin A in the diet. (G) FACS plots (left panels) and bar graphs (right panels) of the percentages of Foxp3+ T cells (CD45+CD4+CD3+) in the SI of mice raised on a synthetic or conventional diet. (H) Concentration of secretory IgA (microgram per milliliter of PBS-dissolved content of the SI) in mice raised on a synthetic or conventional diet, as analyzed by ELISA. Five or six animals were used per group in two separate experiments. *p , 0.05, **p , 0.01, ***p , 0.005. The Journal of Immunology 2175 and divided into three equal parts from the proximal to distal side to vitamin A–converting enzymes, as well as fewer intestinal regula- determine whether differences were present along the gut axis. tory T cells and diminished production of luminal IgA. Mice raised on a synthetic diet showed significantly reduced Transient dietary changes can modulate the mucosal immune aldh1a1 mRNA expression in the proximal part of the SI compared system with mice receiving a standard conventional diet (Fig. 1B). More- over, ALDH activity in CD103+ DCs of the MLN was also sig- We next investigated whether the mucosal immune system could be nificantly reduced in mice that received a synthetic diet (Fig. 1C). influenced by transient changes in the diet. Therefore, mice were The percentages of CD103+ versus CD1032 DCs, as well as the raised on a synthetic diet until 10 wk of age, switched to a con- ALDH activity in CD1032 MLN DCs, were not different between ventional diet for 3 wk, and compared with mice that were kept on the diet groups, pointing to ALDH activity in CD103+ DCs as a synthetic diet. After the dietary change, mRNA expression of selective effect of the different diets (Fig. 1D, 1E). Moreover, to aldh1a1 was increased significantly in the proximal part of SIs + demonstrate that ALDH activity of CD103 DCs did not depend on from mice that were switched to the conventional diet (Fig. 2A). possible differences in dietary vitamin A levels, we tested two In addition, ALDH activity of CD103+ DCs was enhanced sig- levels of vitamin A in the synthetic diet. We raised mice on a nificantly in the MLNs of these mice (Fig. 2B). These data control level of vitamin A (4000 IU/kg) or a high level of vitamin A demonstrate that switching to a conventional diet enhances vita- (20,000 IU/kg). As shown in Fig. 1F, we did not observe any dif- min A metabolism in IECs, leading to more tolerogenic DCs ference in ALDH activity in CD103+ DCs in mice that received a within the intestine. diet with different vitamin A levels, demonstrating that high levels In a reciprocal type of experiment in which we switched adult of dietary vitamin A were not responsible for the differences in Downloaded from mice that were raised on a conventional diet to a synthetic diet, ALDH activity in CD103+ DCs. Because CD103+ DCs are crucial for the induction of regulatory T cells via their production of RA, we measured decreased aldh1a1 mRNA expression in the we examined the percentages of Foxp3+ T cells in the SIs of these proximal part of the SIs compared with mice that were main- mice. Indeed, mice on a synthetic diet showed a reduced percentage tained on the conventional diet (Fig. 2C). In addition, ALDH + of regulatory T cells within the CD4+ T cell population compared activity of CD103 MLN DCs was reduced in the switched mice

with mice fed conventional chow (Fig. 1G). In addition, when we compared with mice that continued to receive a conventional http://www.jimmunol.org/ analyzed the levels of IgA within the content of the SIs, signiﬁ- diet (Fig. 2D). cantly lower IgA concentrations were detected in mice fed the In conclusion, we demonstrate that temporary dietary adjust- synthetic diet (Fig. 1H). Together, these data demonstrate that mice ments can modulate the functionality of the mucosal immune fed the synthetic AIN-93M diet have reduced expression levels of system. by guest on September 27, 2021

FIGURE 2. Temporary dietary changes can affect vitamin A metabolism within the SI. (A and B) C57BL/6 mice were raised on a synthetic AIN-93M diet until 10 wk of age, after which they were switched to a conventional diet or remained on the synthetic diet for 3 wk. (C and D) Additionally, C57BL/6 mice, raised on a conventional diet until 17 wk of age, were switched to a synthetic AIN-93M diet or remained on the conventional diet for 5 wk. In (A) and (C), expression levels of aldh1a mRNA in proximal, middle, and distal parts of the SI were determined by real-time PCR, upon normalization to Ubiquitin and Cyclo mRNA levels. At the end of the experiment, relative mRNA expression levels of proximal SI of mice that received a conventional diet were set at 1.0. In (B) and (D), ALDH activity was measured by ﬂow cytometry in MLN CD103+CD11c+ DCs using the ALDEFLUOR assay, in the absence or presence of ALDH inhibitor DEAB. Six animals were used per group. *p , 0.05, **p , 0.01, ***p , 0.005. 2176 DIETARY FIBERS MODULATE THE MUCOSAL IMMUNE SYSTEM

SCFAs and the aryl hydrocarbon receptor induce vitamin A of zymosan on aldh1a1 expression (Fig. 3F). To further focus on metabolism in a small IEC line microorganism-derived products present within the intestines, we Because our data indicate that the synthetic diet has a reduced used different SCFAs (sodium butyrate, sodium acetate, and so- capacity to induce a tolerant mucosal immune system, we wanted to dium propionate), which are produced upon fermentation of diet- derived fibers, in our culture system. Of the different SCFAs determine which dietary components are responsible for the in- tested, only sodium butyrate led to significantly higher mRNA duction of RALDH1 in small IECs. We tested various dietary levels of the vitamin A–metabolizing enzyme aldh1a1 upon 24-h components that are differentially expressed in the two distinct stimulation of the epithelial cell line (Fig. 3G). diets and that might have an effect on vitamin A metabolism in In sum, of the various molecules tested, sodium butyrate and epithelial cells. Therefore, we set up an in vitro system and made natural Ahr ligands were able to induce aldh1a1 expression in small use of a mouse small IEC line, mICcl2 (27). First, we tested the IECs in culture. effect of dietary conjugated linoleic acid, which was shown to affect vitamin A levels within the liver (34). However, conjugated High-fiber diet induces vitamin A metabolism linoleic acid did not affect aldh1a1 mRNA expression compared Because SCFAs are produced upon digestion of fiber by intestinal with its control, oleic acid (Fig. 3A). In addition, incubation of the microorganisms, we examined whether dietary fiber could spe- epithelial cell line with 25-hydroxycholesterol, which can be cifically induce vitamin A metabolism in small IECs in vivo in a found in the diet as a result of oxidation of cholesterol, also did controlled experiment in which only dietary fiber content was not alter aldh1a1 mRNA levels in the epithelial cell line (Fig. 3B). varied. Therefore, mice were raised on a synthetic diet containing Furthermore, we tested aryl hydrocarbon receptor (AhR) ligands, 3.2% fiber that was followed, for 2 wk, by a synthetic diet con- Downloaded from which are formed during digestion of cruciferous vegetables, be- taining no fiber (0%) or a diet with a high fiber level (35%). As cause their importance in mucosal immune regulation and devel- shown previously, high-fiber diets led to the increased production of opment is well known (2, 35). Although the synthetic AhR ligand different SCFAs by microbiota in the gut (18, 36). Analysis of SIs TCDD did not exert any effect (Fig. 3C), the natural AhR ligand revealed a significantly higher expression of aldh1a1 mRNA in DIM was able to increase aldh1a1 mRNA expression after 24 h of mice receiving a high-fiber diet compared with mice receiving a treatment (Fig. 3D). However, when we analyzed whether the no-fiber diet (Fig. 4A), demonstrating the ability of dietary fiber to http://www.jimmunol.org/ expression of aldh1a1 was changed in the SI of AhR-knockout induce vitamin A metabolism in IECs. Furthermore, to confirm mice, we did not observe any difference compared with control that SCFAs are responsible for increased RALDH1 levels when mice (Fig. 3E), thereby excluding a central role for this ligand– mice received a high-fiber diet, sodium butyrate was supplied in receptor pair in vivo. the drinking water for 3 wk. Subsequent analysis revealed that To further examine whether fungal-derived ligands, which are sodium butyrate increased aldh1a1 mRNA expression levels in the present within the intestinal lumen, affect aldh1a1 expression, we proximal part of the SI (Fig. 4B). incubated the epithelial cell line with zymosan, a b-glucan present To gain more insight in the mechanisms by which SCFAs influence in the cell wall of fungi. However, we did not observe any effects epithelial cells, we performed the same fiber diet experiments with by guest on September 27, 2021

FIGURE 3. Expression of aldh1a1 enzyme in mICcl2 epithelial cells can be induced by a natural Ahr ligand and SCFA butyrate. The mICcl2 small IEC line was stimulated for 24 h with various dietary- or microbiota-derived products and analyzed for aldh1a1 mRNA expression of conjugated linoleic acid (CLA) and oleic acid (OLA) as an appropriate control (A), 25-hydroxycholesterol (B), synthetic Ahr ligand TCDD (C), natural Ahr ligand DIM (D), fungal cell wall component zymosan (F), and the SCFAs sodium acetate (SA), sodium butyrate (SB), and sodium propionate (SP) (G). The medium control was set at 1.0 for all in vitro stimulations. (E) Expression levels of aldh1a1 in proximal, middle, and distal parts of SIs from WT and Ahr-knockout mice; mRNA levels were set at 1.0 in the proximal SI of WT mice. The expression levels of aldh1a1 mRNA were analyzed by qPCR and normalized to those of Ubiquitin and Cyclo. In vitro experiments were repeated three to seven times; in vivo experiments included six mice per group. **p , 0.01, ***p , 0.005. The Journal of Immunology 2177 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. High-fiber diet induces vitamin A metabolism independently of GPR signaling. (A–D) Mice were raised on a synthetic AIN-93 control diet (3.2% fiber) until 8–10 wk of age. Subsequently, diets were switched to ones that contained no fiber and high levels of fiber (35%) for 2 wk. WT C57BL/6 mice (A), GPR1092/2 mice (C), and GPR432/2 mice (D). (B) Moreover, WT C57BL/6 mice received sodium butyrate (SB; 100 mM) for 3 wk in their drinking water. SIs were collected, and expression levels of aldh1a1 in proximal, middle, and distal parts were measured; mRNA levels were set at 1.0 in the proximal SI part of WT mice that received the no-fiber diet. Five or six animals were used per group. (E) mICcl2 were analyzed for aldh1a1 expression after a 30-min incubation with pertussis toxin (PT) before SB stimulation for 24 h. Stimulation of mICcl2 cells with 4-CMTB were analyzed for aldh1a1 (F) and nlpr3 (G). mRNA expression levels of aldh1a1 and nlpr3 were normalized to those of Ubiquitin and Cyclo. In vitro experiments were repeated three to eight times; in vivo experiments used six mice per group. *p , 0.05, **p , 0.01.

GPR43- and GPR109-knockout mice, because SCFAs can mediate data suggest that, in the absence of GPR43, aldh1a1 expression is their effects via various GPCRs (8, 37). In the absence of GPR109, induced independently of fiber consumption and is likely to be aldh1a1 expression still increased upon consumption of high-fiber regulated via other mechanisms. In addition, when we stimulated diet compared with mice that received a diet that lacked fiber, the epithelial cell line with sodium butyrate after it was pre- indicating that induction of aldh1a1 expression can occur inde- incubated with pertussis toxin, which inhibits Gi-coupled GPCR pendently of GPR109 (Fig. 4C). In contrast, aldh1a1 expression signaling, aldh1a1 mRNA expression could still be induced was not significantly induced in GPR43-knockout mice upon (Fig. 4E). To further explore the role of GPR43, we directly consumption of a high-fiber diet, suggesting a possible involve- stimulated mICcl2 cells with the GPR43 agonist 4-CMTB. First, to ment for this receptor (Fig. 4D). However, compared with WT confirm that 4-CMTB stimulation worked, we analyzed expres- mice (Fig. 4A), expression of aldh1a1 within the SIs of mice sion of nlpr3 as a positive control, because we demonstrated re- receiving a no-fiber diet was higher in GPR432/2 mice. These cently that SCFA binding to GPR43 leads to NLPR3 inflammasome 2178 DIETARY FIBERS MODULATE THE MUCOSAL IMMUNE SYSTEM activation in IECs (38). Indeed, we observed a significant induction of nlpr3 after 4-CMTB stimulation of the epithelial cells (Fig. 4F). However, when we analyzed the expression of aldh1a1, we did not see any differences after 4-CMTB stimulation, whereas sodium butyrate significantly induced aldh1a1 expression (Fig. 4G). These results demonstrated that dietary fiber was able to induce the expression of RALDH1 in the intestines, which was not dependent on GPR signaling. Specific HDAC inhibition induces vitamin A metabolism in a small IEC line In addition to their ability to signal via GPCRs, SCFAs can act as HDAC inhibitors. Because the DNA is wrapped around histones within the nucleus of the cell, acetyl and deacetyl transferases can manipulate the chromatin conformation, leading to more open chromatin and thus active or silenced gene expression, respectively (39). Therefore, HDAC inhibitors can have a direct effect on the expression of genes that are regulated by the specific HDACs. To Downloaded from test whether SCFAs induce aldh1a1 levels via the inhibition of HDAC, we stimulated mICcl2 cells with a general HDAC inhibitor, TSA (Fig. 5A). Indeed, aldh1a1 expression was strongly induced upon culture with TSA. Moreover, no additive effect was found when we stimulated the epithelial cell line with sodium butyrate

and TSA at the same time (Fig. 5B). These results indicate that http://www.jimmunol.org/ SCFAs induce expression of the vitamin A–converting enzyme FIGURE 5. Expression of aldh1a1 enzyme in mICcl2 epithelial cells can be induced by specific HDAC inhibition. (A) aldh1a1 levels of mIC cells RALDH1 in IECs by inhibiting HDAC activity. To further define cl2 stimulated with the HDAC inhibitor TSA, MS275, M344, or Droxinostat which HDAC could be involved in regulating RALDH enzyme for 24 h. (B) aldh1a1 levels of mICcl2 cells stimulated with sodium bu- expression levels, we stimulated the epithelial cell line with a tyrate (SB), TSA, and SB+TSA. (C) villin levels of mICcl2 cells stimulated more specific HDAC inhibitor, MS275, which inhibits HDAC1 with SB, TSA, MS275, M344, or Droxinostat. The medium control was set and HDAC3. After stimulation with this specific HDAC inhibitor, at 1.0 for all in vitro experiments. All mRNA expression levels of aldh1a1 we observed a significant induction of aldh1a1 expression in the and villin were analyzed by qPCR and normalized to those of Ubiquitin epithelial cells (Fig. 5A). These data implicate a role for HDAC1 and Cyclo. In vitro data are representative of four to seven experiments. or HDAC3 in controlling vitamin A metabolism in IECs. How- **p , 0.01, ***p , 0.005. ns, not significant. by guest on September 27, 2021 ever, when we stimulated the epithelial cell line with Droxinostat, which specifically inhibits HDAC3, HDAC6, and HDAC8, we dominant in the mice fed a conventional diet. This lactate- observed only a slight increase in aldh1a1 expression. Stimulation producing genus was shown to indirectly stimulate bacterial bu- of epithelial cells with M344, which specifically inhibits HDAC1 tyrate production (40, 41). In addition, the Bacteroidetes phylum, and HDAC6, resulted in a much higher expression of aldh1a1, of which some members are able to produce SCFAs themselves similar to the effects of TSA or MS275. These data imply that (40, 42), was reduced in mice fed a synthetic diet (Fig. 6B). HDAC1 is involved in controlling aldh1a1 expression in IECs. As Therefore, these bacteria species could be involved in the pro- a control, we measured expression of the actin-binding protein duction of SCFAs in mice receiving a conventional diet. When villin, which is highly expressed in epithelial cells. When epi- two isomers of lactic acid and SCFAs were measured in the SI, thelial cells were stimulated with sodium butyrate or one of the significantly lower levels were detected in mice fed a synthetic HDAC inhibitors, we did not observe any differences in villin diet versus a conventional diet (Fig. 6C, 6D). D-Lactic acid and expression (Fig. 5C). These results demonstrate that HDAC in- propionic acid were even below the detection limit in the content hibition does not generally increase gene expression and that the of the SI of mice fed a synthetic diet (Fig. 6C, 6D). Additionally, induction of aldh1a1 expression after HDAC inhibition is specific. an increased level of Erysipelotrichia was observed in mice that In sum, our results indicate that dietary fiber is able to induce received a synthetic diet (Fig. 6B). These data are in agreement vitamin A metabolism in small IECs via the inhibition of HDAC. with earlier studies in which low-fiber diets led to an increase in Firmicutes, mainly Erysipelotrichia (36). Furthermore, the syn- Changes in the distribution of microbiota in the SI upon dietary thetic diet led to a clear increase in the mucus-degrading bacterial adjustment genus Akkermansia (phylum Verrucomicrobia). Because microorganisms present in the intestines are critical for the In conclusion, we showed that the microbiota composition in the production of SCFAs, we analyzed the composition of the SI changed upon dietary adjustments. These microbial alterations microbiota within the SIs of mice that were raised on a conventional affected the production of SCFAs by the microbiota in the SIs, or a synthetic diet. To determine microbial diversity in these having an effect on vitamin A metabolism within IECs. samples, a barcoded amplicon pyrosequencing method was used (31). Indeed, clear differences could be observed between the two Discussion diet groups (Fig. 6A). Sequences affiliated with the butyrate- Although the importance of tolerogenic CD103+ DCs for the producing family Lachnospiraceae (phylum Firmicutes, order maintenance of mucosal homeostasis is well established, the exact Clostridiales) were less dominant in the mice fed a synthetic diet mechanism by which the differentiation of these cells is regulated (Fig. 6B). Similarly, sequences affiliated with the genus Lacto- is not clear. It is described that the phenotype of these DCs de- bacillus (phylum Firmicutes, order Lactobacillales) were more pends on the production of RA by epithelial cells (23, 24, 26); The Journal of Immunology 2179 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Composition of the microflora within the SI of mice raised on conventional and synthetic diets. The composition of the microflora within the SIs of mice, raised on a conventional or synthetic diet, was analyzed with the use of a barcoded amplicon pyrosequencing technique. (A) Relative abundance of bacterial classes detected in each stool sample, represented as a heat map. (B) Measurements of specific bacteria in mice fed a conventional diet or a synthetic diet. The concentrations of two isomers of lactic acid (D-lactic acid and L-lactic acid) (C) and SCFAs (acetic acid, propionic acid, and butyric acid) (D) were measured within the content of the SI. Five or six animals were used per group. *p , 0.05, **p , 0.001. N.D., not detected. 2180 DIETARY FIBERS MODULATE THE MUCOSAL IMMUNE SYSTEM however, it is not understood how the production of RA by epi- Moreover, we demonstrated that differences in diet resulted in thelial cells themselves is controlled. Although we saw in previous alterations in the composition of the microbiota. Whether these studies that ALDH activity of CD103+ DCs is ablated in mice that changes directly or indirectly affect the mucosal immune system are deficient for vitamin A, aldh1a1 mRNA expression in the SIs cannot be stated at this point; however, we showed that SCFA- did not depend on the intake of vitamin A (21). In this study, we producing taxa were reduced in the SI of mice that received a demonstrated that additional dietary compounds are crucial for the synthetic diet. These bacterial changes led to lower levels of SCFAs regulation of vitamin A metabolism in IECs, which leads to in- compared with conventionally fed mice. creased ALDH activity in CD103+ MLN DCs and a concomitant In addition, it was shown that obese humans and mice have an increase in regulatory T cells within the intestines and production altered gut microbiota, with decreased levels of Bacteroidia bac- of luminal IgA. The responsible dietary compound is fiber, which teria (16, 48); these results were similar to our mice that received a gives rise to SCFAs upon digestion by the microflora. Moreover, synthetic diet. Furthermore, we demonstrated that bacteria of the SCFAs, especially butyrate, were able to induce vitamin A genus Erysipelotrichia (phylum Firmicutes) were enhanced sig- metabolism in the small IEC line mICcl2. Our data confirmed nificantly in mice that received a synthetic diet, correlating with results from an earlier study in which mice that received a diet mice that received a high-fat diet or mice with colorectal cancer supplemented with cycloinulooligosaccharides had increased (16, 49). Moreover, previous studies demonstrated that microbiota levels of SCFAs and higher concentrations of IgA measured in the diversity is diminished when a low-fiber diet is consumed. Ad- content of the SI (43). Recently, we also demonstrated the role of ditionally, they showed that microbiota in mice fed a low-fiber diet SCFAs in a model of food allergy and found that SCFAs, spe- was dominated by the phylum Firmicutes, mainly Erysipelo- cifically acetate and butyrate, added to the drinking water in- trichia, whereas the levels of Bacteroidia were reduced, resem- Downloaded from creased tolerogenic DCs and tolerogenic T cells in the MLN (44). bling our results in mice fed a synthetic diet and that most likely Furthermore, it was shown that SCFAs, mainly acetate and buty- consumed less fiber than mice fed a conventional diet (36). Fur- rate, were enhanced in the cecum and colon of mice that received thermore, in mice fed a synthetic diet, the levels of Akkermansia a high-fiber diet (18, 36). We observed increased vitamin A (phylum Verrucomicrobia), which can be found in the inner mu- metabolism in mice that received a high-fiber diet. By focusing on cous layer where they degrade mucus and, thereby, stimulate

the effects within the SIs, we could determine changes in the mucus production by goblet cells (50), were significantly lower. http://www.jimmunol.org/ microbiota and SCFA levels that impacted the epithelial cells and Moreover, the intestinal mucous layer is altered in a number of the mucosal immune system. In line with our data is the report that intestinal diseases linked to a deficiency in dietary fiber. stromal cells in the lamina propria of the SIs can also produce RA, A balanced symbiotic relationship between host and microbiota which is dependent on the presence of microbiota in the gut. Like exists during intestinal homeostasis; however, gut dysbiosis can IECs, these RA-producing stromal cells are in close contact with lead to a variety of diseases (13). The best-known therapeutic CD103+ DCs. Together, these data suggest that, in addition to approaches to restore a healthy gut microflora are antibiotics, IECs, stromal cells might play a role in the induction of tolero- prebiotics, and probiotics, (51, 52). Particular combinations of genic DCs (45). However, it remains to be seen whether these bacteria might be needed to successfully restore symbiosis (15, 51, stromal cells are imprinted directed by luminal contents or 53). by guest on September 27, 2021 whether they depend on IEC-derived factors. However, the im- In conclusion, we showed that dietary factors are able to induce portance of the induction of these tolerogenic DCs was further aldh1a1 expression in IECs. Subsequently, these epithelial cells highlighted by Magnusson et al. (46) recently; they found that are able to induce tolerogenic CD103+ DCs through their pro- patients with ulcerative colitis have reduced Aldh1+ cells (mac- duction of RA. In turn, these DCs produce RA themselves and, rophages and DCs) in the colon, regardless of their inflammation thereby, are able to induce regulatory T cells and IgA-producing status. B cells. Furthermore, SCFAs are able to induce aldh1a1 expres- Although our data from GPR43- and GPR109-knockout mice did sion in IECs through inhibition of HDAC. The availability of not demonstrate a central role for the induction of vitamin A substrates determines the composition of the microbiota and the metabolism upon high fiber intake, based on these data we cannot production of SCFAs, because these are produced upon digestion fully exclude a role for GPCRs in the induction of vitamin A of the substrates by the microbiota. These findings reveal a deli- metabolism within IECs. Various GPCR receptors are able to bind cate mechanism that controls the mucosal immune system and that the various SCFAs with different affinity (8, 37). Therefore, the can have important implications for the prevention and treatment lack of only one receptor might not lead to differences in SCFA of immune-mediated chronic diseases. signaling because of receptor redundancy. To rule out the role of GPCR receptors, triple-knockout mice for the main GPCRs that Acknowledgments bind SCFA (GPR41, GPR43, and GPR109) may need to be an- We thank C. Prins, H. van der Laan, and E. van Gelderop (VU University, alyzed. However, when we inhibited G protein signaling during Amsterdam, the Netherlands) for animal care. sodium butyrate stimulation in vitro, induction of aldh1a1 expression still occurred, showing no role for G protein–mediated Disclosures signaling in controlling vitamin A metabolism in IECs. Fur- The authors have no financial conflicts of interest. thermore, when we stimulated the IEC line with a specific GPR43 agonist, no induction of aldh1a1 expression was observed. References In addition to binding to GPCRs, SCFAs can act as HDAC 1. Hooper, L. V. 2011. You AhR what you eat: linking diet and immunity. Cell 147: 489–491. inhibitors. We were able to show in vitro that inhibition of HDAC 2. Kiss, E. A., C. Vonarbourg, S. Kopfmann, E. Hobeika, D. Finke, C. Esser, and activity resulted in increased expression of aldh1a1, and our data A. Diefenbach. 2011. Natural aryl hydrocarbon receptor ligands control organ- implicated HDAC 1 in the control of aldh1a1 expression levels in ogenesis of intestinal lymphoid follicles. Science 334: 1561–1565. IECs. In line with our results, it was demonstrated recently that 3. Spencer, S. P., C. Wilhelm, Q. Yang, J. A. Hall, N. Bouladoux, A. Boyd, T. B. Nutman, J. F. Urban, Jr., J. Wang, T. R. Ramalingam, et al. 2014. Adap- other IEC lines and organoids induce their aldh activity after tation of innate lymphoid cells to a micronutrient deficiency promotes type 2 SCFA stimulation (47). barrier immunity. Science 343: 432–437. The Journal of Immunology 2181

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