Dietary Palmitic Acid and Its Metabolism Regulation of Intestinal

Home , Palmitic acid

Regulation of Intestinal IgA Responses by Dietary Palmitic Acid and Its Metabolism Jun Kunisawa, Eri Hashimoto, Asuka Inoue, Risa Nagasawa, Yuji Suzuki, Izumi Ishikawa, Shiori Shikata, Makoto Arita, This information is current as Junken Aoki and Hiroshi Kiyono of September 29, 2021. J Immunol 2014; 193:1666-1671; Prepublished online 16 July 2014; doi: 10.4049/jimmunol.1302944

http://www.jimmunol.org/content/193/4/1666 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2014/07/16/jimmunol.130294 Material 4.DCSupplemental http://www.jimmunol.org/ References This article cites 42 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/193/4/1666.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

by guest on September 29, 2021 • No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulation of Intestinal IgA Responses by Dietary Palmitic Acid and Its Metabolism

Jun Kunisawa,*,†,‡,x,{ Eri Hashimoto,*,† Asuka Inoue,‡,‖ Risa Nagasawa,*,† Yuji Suzuki,† Izumi Ishikawa,† Shiori Shikata,*,† Makoto Arita,#,**,1 Junken Aoki,‖,†† and Hiroshi Kiyono†,‡,††,‡‡,xx

Enhancement of intestinal IgA responses is a primary strategy in the development of oral vaccine. Dietary fatty acids are known to regulate host immune responses. In this study, we show that dietary palmitic acid (PA) and its metabolites enhance intestinal IgA responses. Intestinal IgA production was increased in mice maintained on a PA-enriched diet. These mice also showed increased intestinal IgA responses against orally immunized Ag, without any effect on serum Ab responses. We found that PA directly stimulates plasma cells to produce Ab. In addition, mice receiving a PA-enriched diet had increased numbers of IgA-producing plasma cells in the large intestine; this effect was abolished when serine palmitoyltransferase was inhibited. These ﬁndings suggest that Downloaded from dietary PA regulates intestinal IgA responses and has the potential to be a diet-derived mucosal adjuvant. The Journal of Immunology, 2014, 193: 1666–1671.

igh levels of IgA are present in the intestine, where they immunosurveillance in the intestine, the primary goal of effective oral protect the host against pathogenic microorganisms by vaccines is the efficient induction of Ag-specific IgA responses (3). H preventing their attachment to and entrance into epithelial An efficient intestinal IgA response requires host-derived factors, http://www.jimmunol.org/ cells, as well as by neutralizing their toxins (1). Some patients with including cytokines (e.g., IL-5, IL-6, IL-10, IL-15, APRIL, BAFF) IgA deficiency show increased susceptibility to infectious pathogens, and chemokines (e.g., CCL25/CCR9) (4, 5), as well as immunologic including Giardia lamblia, Campylobacter, Clostridium, Salmonella, cross-talk with environmental factors (e.g., commensal bacteria and and rotavirus (2). Given the immunologic importance of IgA in dietary materials) (6). Indeed, germ-free mice have decreased intestinal IgA responses because of the immature structure of Peyer’s patches (PPs) and isolated lymphoid follicles (7, 8). We recently *Laboratory of Vaccine Materials, National Institute of Biomedical Innovation, identified a unique subset of intestinal IgA-producing plasma cells Osaka 567-0085, Japan; †Division of Mucosal Immunology, Department of Microbiol-

ogy and Immunology, The Institute of Medical Science, University of Tokyo, Tokyo (PCs) in the murine intestine; these cells expressed CD11b, re- by guest on September 29, 2021 108-8639, Japan; ‡International Research and Development Center for Mucosal Vac- quired microbial stimulation and mature PP structure, proliferated cines, The Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; xDepartment of Microbiology and Immunology, Kobe University School of Med- vigorously, and produced high amounts of IgA (9). icine, Kobe 650-0017, Japan; {Graduate School of Pharmaceutical Sciences and In addition to commensal bacteria, nutritional molecules, such ‖ Graduate School of Dentistry, Osaka University, Osaka 565-0871, Japan; Labo- as vitamins, are essential for the development, maintenance, and ratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceu- tical Sciences, Tohoku University, Sendai 980-8578, Japan; #Department of regulation of intestinal immune responses (6, 10, 11). Therefore, Health Chemistry, Graduate School of Pharmaceutical Sciences, University of nutritional deficiencies and inappropriate dietary intake increase Tokyo, Tokyo 113-0033, Japan; **Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, the risk for infectious, allergic, and inflammatory diseases (12, Japan; ††Core Research for Evolutional Science and Technology, Japan Science 13). Among various dietary factors, oils are known to influence host ‡‡ and Technology Agency, Saitama 332-0012, Japan; Department of Medical Ge- immune function and inflammatory responses (14–16). Overnutri- nome Science, Graduate School of Frontier Science, University of Tokyo, Chiba 277- 8561, Japan; and xxGraduate School of Medicine, University of Tokyo, Tokyo 113- tion due to a high-fat diet leads to the development of inflammation 0033, Japan in adipose tissue, which is frequently associated with obesity and 1Current address: Laboratory for Metabolomics, RIKEN Center for Integrative Med- atherosclerosis (17). Recent findings suggest that, in addition to the ical Sciences, Yokohama, Kanagawa, Japan. quantity of oil ingested, the fatty acid (FA) composition of the oil is Received for publication November 1, 2013. Accepted for publication June 11, 2014. an important factor in various immunologic and inflammatory This work was supported by grants from the Science and Technology Research conditions (14–16). Dietary oil is generally composed of long-chain Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry (to J.K.); the Program for Promotion of Basic and Applied Research for Innovations in Bio- saturated FAs (e.g., C16:0 palmitic acid [PA] and C18:0 stearic oriented Industry (to J.K.); the Ministry of Education, Culture, Sports, Science, and acid) and mono- or polyunsaturated FAs (PUFAs; e.g., C18:1 oleic Technology of Japan (to J.K., M.A., J.A., and H.K.); the Ministry of Health and a a Welfare of Japan (to J.K. and H.K.); the Yakult Bio-Science Foundation (to J.K.); the acid, C18:2 linoleic acid, and C18:3 -linolenic acid). -Linolenic Ono Medical Research Foundation (to J.K.); the Japan Science and Technology acid and linoleic acid are precursors of v3 and v6 PUFAs, re- Agency, Core Research for Evolutional Science and Technology (to J.A. and H.K.); spectively; v3 FAs are metabolized into anti-inflammatory mol- and Precursory Research for Embryonic Science and Technology (to M.A.). ecules, whereas v6 FAs are converted into proinflammatory lipid Address correspondence and reprint requests to Dr. Jun Kunisawa, Laboratory of a Vaccine Materials, National Institute of Biomedical Innovation, 7-9-8 Asagi Saito, mediators (16, 18). Therefore, the ratio of -linolenic acid/linoleic Osaka 567-0085, Japan. E-mail address: [email protected] acid in dietary oils is thought to determine the onset of various The online version of this article contains supplemental material. immunologic conditions. In addition to modulating the v3–v6PUFA Abbreviations used in this article: CT, cholera toxin; FA, fatty acid; iLP, intestinal balance, saturated FAs, such as PA, stimulate host immune responses lamina propria; PA, palmitic acid; PC, plasma cell; PP, Peyer’s patch; PUFA, poly- by promoting the production of proinflammatory cytokines, in- unsaturated fatty acid; S1P, sphingosine 1 phosphate; SPT, serine palmitoyltransferase. cluding IL-6 and TNF-a (19, 20). Although these proinflammatory Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 cytokines are prerequisite factors for the efficient induction of IgA www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302944 The Journal of Immunology 1667 responses (5), the immunologic function of dietary PA in the control fluorescent Abs specific for B220, IgG1, IgA (all from BD Biosciences), of IgA production remained to be investigated. In this study, we CD19, and CD138 (BioLegend, San Diego, CA). Forward scatter–height show that PA-enriched diets enhance intestinal IgA responses both and forward scatter–area discrimination was used to exclude doublet cells, and Via-Probe Cell Viability Solution (BD Biosciences) was used to dis- directly and through their metabolic pathways. criminate between dead and living cells. For the BrdU-uptake assay, mice received 1 mg BrdU i.p., and the BrdU signal was detected according to Materials and Methods the manufacturer’s protocol (BD Biosciences) (9). Concentration-matched Mice isotype Abs were used as negative controls. Flow cytometric analysis and cell sorting were carried out using FACSCanto II and FACSAria (BD Female BALB/c, C3H/HeJ, and C3H/HeN mice were purchased from Biosciences), respectively. We confirmed that cell purity was ∼95%. CLEA Japan (Tokyo, Japan). Chemically defined AIN-93M–based diets containing soybean, palm, or coconut oil or soybean oil plus supplemental In vitro culture of PCs with PA purified PA were from Oriental Yeast (Tokyo, Japan). All mice were pro- Stock solutions containing 5 mM PA and 10% BSA were prepared as vided with a sterile diet and water ad libitum. To inhibit serine palmi- previously reported (23). Purified IgA+ B2202 PCs isolated from the iLP toyltransferase (SPT) activity, mice were treated with myriocin (1.0 mg/kg (104 cells/well) or CD19+ CD138+ PCs isolated from the spleen (105 cells/ i.p. daily; Sigma-Aldrich, St. Louis, MO) for 4 d (21). All mice were well) were cultured with 10 or 100 mM PA for 96 h. The amount of IgA maintained in the experimental animal facility at the University of Tokyo (for iLP) or IgG (for spleen) in the culture supernatant was determined by and National Institute of Biomedical Innovation; the experiments were ap- ELISA, as described earlier. proved by the Animal Care and Use Committee of each institute and conducted in accordance with their guidelines. Measurement of PA in the serum and intestinal tissues Cell isolation Lipids were extracted from serum and intestinal tissues by using

chloroform–methanol and chloroform solutions. The specimens were Downloaded from To isolate mononuclear cells from PPs, we stirred intestinal tissues in RPMI dried in nitrogen gas and dissolved in 0.4 M potassium methoxide in 1640 medium containing 2% FCS and 0.5 mg/ml collagenase (Wako, methanol and 14% boron trifluoride in methanol. The FA concentrations in Osaka, Japan). Cells were isolated from the intestinal lamina propria (iLP) the solutions were measured (SRL, Tokyo, Japan) using gas chromatog- as previously described (9, 22). Briefly, PPs were removed, and small and raphy (model GF 17A; Shimazu, Kyoto, Japan). large intestines were cut into 2-cm pieces, which were stirred in RPMI 1640 containing 1 mM EDTA and 2% FCS. Then the tissues were stirred Statistics in collagenase for 15 min three times (small intestine, 0.8 mg/ml; large intestine, 1.6 mg/ml) before undergoing discontinuous Percoll gradient Experimental groups were compared using the Mann–Whitney U test http://www.jimmunol.org/ centrifugation. Lymphocytes were isolated at the interface between the 40 (GraphPad, San Diego, CA). and 75% layers. Oral immunization Results Mice maintained on a diet containing palm oil show enhanced Mice received sodium bicarbonate to neutralize gastric acid before oral intestinal IgA production immunization (9, 22). Thirty minutes later, the mice were immunized orally concurrently with 1 mg OVA (Sigma-Aldrich) and 10 mg cholera To examine whether dietary PA affects intestinal IgA production, toxin (CT; List Biological Laboratories, Campbell, CA). This oral- we maintained mice on a diet containing 4% soybean (control) or immunization procedure was conducted on days 0, 7, and 14. palm (PA-rich) oil for 2 mo (Fig. 1A) and measured the amounts of by guest on September 29, 2021 Detection of total Ig by ELISA and OVA-specific Ab responses fecal IgA. Intestinal IgA production was higher in the mice that by ELISPOT assay received palm oil than in those given soybean oil (Fig. 1B). In contrast, the amounts of serum IgG and IgA were similar between Total Ig levels in serum and fecal extracts were determined by ELISA, as previously described (9, 22). To measure Ab concentration, purified murine the soybean and palm oil groups (Fig. 1B, Supplemental Fig. 1). isotype-specific Abs (BD Biosciences, San Jose, CA) were used as Palm oil is high in both PA and oleic acid (Fig. 1A), but mice standards for quantification. For the detection of OVA-specific Abs and maintained on a diet containing rapeseed oil, which contained a Ab-forming cells, fecal extracts and iLP were prepared 7 d after the final similar amount of oleic acid as that present in the palm oil, immunization. Standard OVA-specific ELISAs and ELISPOT assays were performed as previously described (9, 22). showed no enhancement of intestinal IgA production (data not shown). Flow cytometry and cell sorting We then considered whether other saturated FAs enhance in- Flow cytometry and cell sorting were performed as previously described (9, testinal IgA production. To this end, we used coconut oil, which 22). Cells were preincubated with anti-CD16/32 Ab and then stained with (like palm oil) is a Palmae plant–based oil but contains large

FIGURE 1. Palm oil enhances intestinal IgA production. (A) FA composition of soybean, palm, and coconut oils. (B) Mice were maintained on a diet containing soybean, palm, or coconut oil for 2 mo. Feces and serum samples were collected for measurement of the amounts of IgA and IgG, respectively (mean 6 1 SD, n = 10/group). 1668 DIETARY PALMITIC ACID IN INTESTINAL IgA PRODUCTION amounts of other saturated FAs, including lauric and myristic acids (Fig. 1A). Unlike palm oil, coconut oil did not alter fecal or serum Ab production in the mice (Fig. 1B). These findings suggest that dietary PA uniquely enhances intestinal IgA production. PA-enriched dietary oils enhance intestinal IgA production and IgA responses against oral vaccine Ag To confirm that PA enhances intestinal IgA production, we added PA to soybean oil to adjust its PA concentration to that of palm oil (Fig. 2A, upper bar). Fecal IgA levels in mice maintained for 2 mo on a diet containing the PA-enriched soybean oil were increased similarly to those of mice fed a palm oil–containing diet, FIGURE 3. Ingestion of PA-enriched oil enhances intestinal IgA re- but serum IgA production was unchanged (Fig. 2A, lower bar, sponses against orally immunized Ag. After mice were maintained for Supplemental Fig. 1). These data suggest that PA is a key FA in 2 mo on a diet containing soybean oil, soybean oil + PA, or palm oil, they were orally immunized with OVA plus CT on days 0, 7, and 14. On day 21, the enhancement of intestinal IgA production. fecal extracts were collected for the determination of OVA-specific intes- We then investigated whether the enhanced production of in- tinal IgA by ELISA. Data are mean 6 1SD(n = 4/group), and similar testinal IgA induced by dietary PA is reflected in the responses to an results were obtained from two independent experiments. *p , 0.05. orally administered vaccine. To this end, we orally immunized mice concurrently with OVA and the mucosal adjuvant CT. In agreement Downloaded from PA directly stimulates intestinal PCs to produce IgA with levels of naturally produced IgA, OVA-specific fecal IgA responses were enhanced in mice maintained on a diet that con- We then examined whether PA directly affected IgA production + tained palm oil (Fig. 3). In addition, similarly increased OVA-specific from PCs. To address this issue, we purified IgA PCs from the IgA production was noted in mice maintained on PA-enriched intestine and cultured them with PA for 4 d. The amount of IgA in soybean oil (Fig. 3). These findings suggest that dietary PA enhanced the intestinal PC culture supernatants increased in a dose-dependent manner (Fig. 5A). ELISPOT assays showed that PA did not increase not only naturally produced intestinal IgA but also Ag-specific http://www.jimmunol.org/ intestinal IgA induced by oral immunization. the number of IgA-forming cells (Fig. 5B), suggesting that PA in- stead enhances Ab production from PCs. In addition, IgG produc- PA content is increased in the intestinal, but not systemic, tion from CD19+ CD138+ PCs from the spleen was increased in the compartment presence of PA (Supplemental Fig. 2), indicating that PA enhances We next measured the amounts of PA in the intestines and serum of Ab production, regardless of the Ig subtype. the mice. PA concentrations in the small and large intestines were PA acts as a ligand for TLR4 (24), prompting our investigation higher in the mice maintained on PA-rich soybean oil compared into whether TLR4 mediated the PA-induced direct activation of with soybean oil alone (Fig. 4). In contrast, PA amounts in serum IgA PCs. To address this issue, we used C3H/HeN and C3H/HeJ

were comparable between groups (Fig. 4). These findings indicate mice. Although TLR4-mediated signaling differs between these by guest on September 29, 2021 that dietary PA affects the amount of PA locally in the intestine two strains because of a spontaneous point mutation in the Tlr4 without any influence on serum PA concentration. gene of the C3H/HeJ mice (25, 26), intestinal PCs from these mouse strains produced identical levels of PA-induced IgA (Fig. 5C). Therefore, the direct effect of PA on IgA production from PCs likely is independent of the TLR4 pathway. The PA-induced increase in IgA PCs in the large intestine is dependent on SPT We used flow cytometry to determine the frequency of IgA+ PCs in the small and large intestines. Although no significant difference between the control and PA-enriched diets was noted in the small intestine, the frequency of IgA+ B2202 PCs was increased in the

FIGURE 4. Increased PA content in the intestine of mice maintained on FIGURE 2. Dietary PA alone is sufﬁcient to enhance intestinal IgA pro- PA-enriched oils. Mice were maintained for 2 mo on a diet containing duction. (A) Puriﬁed PA was added to soybean oil to achieve the same PA soybean oil, with or without PA, and small and large intestinal tissues and content as that in palm oil. (B) Mice were maintained for 2 mo on a diet con- serum were collected for measurement of their PA contents. The graphs taining soybean oil, with or without supplemental PA. The IgA levels of fecal show data from individual mice, and the horizontal lines indicate the extracts were measured (mean 6 1SD,n = 16/group). means; similar results were obtained from two independent experiments. The Journal of Immunology 1669

FIGURE 5. PA directly stimulates IgA-producing PCs via a TLR4-independent mechanism. IgA+ B2202 PCs were isolated from the small intestine of BALB/c (A and B) and C3H/HeN or C3H/HeJ mice (C) and cultured with 10 or 100 mM(A) or 100 mM(B and C) PA. The amount of IgA in the culture supernatant (A and C) and the number of IgA+ Ab–forming cells (IgA+-AFCs) (B) were determined by ELISA and ELISPOT, respectively. The graphs show data from individual samples, and the horizontal lines indicate the means; similar results were obtained from two independent experiments (A and C). Data in (B) are mean 6 1SD(n = 10/group from two separate experiments).

large intestine of the PA-enriched diet group (Fig. 6A). Similarly, lipids all known to promote cell proliferation, survival, and trafﬁcking Downloaded from the number of OVA-speciﬁc IgA-forming cells was increased in (27, 28). Therefore, we supposed that these PA-derived sphingolipids the large, but not small, intestine of mice receiving oral immu- might be involved in the regulation of intestinal IgA PCs in the large nization and the PA-enriched diet (Fig. 6B). In agreement with intestine. To test this possibility, mice were treated with myriocin, the lack of a PA-associated effect on small intestinal IgA, B cell an inhibitor of SPT, which is a key enzyme in the conversion of PA differentiation into IgA+ cells in the PPs, a lymphoid tissue for into sphingolipids (Fig. 6C). The PA-mediated increase in IgA PCs the initiation of small intestinal IgA responses (3), was unchanged did not occur in the large intestine of mice that received myriocin http://www.jimmunol.org/ in mice maintained on a PA-enriched diet (Supplemental Fig. 3). (Fig. 6D), and myriocin had little effect on the number of IgG1+ PA can be converted into sphingolipids, including ceramide, cells in the spleen (Supplemental Fig. 4), suggesting that SPT ac- sphingosine, and sphingosine 1 phosphate (S1P) (Fig. 6C); all of these tivity is required for this effect on large intestinal IgA PCs. by guest on September 29, 2021

FIGURE 6. SPT is required for the increased number of IgA PCs in the large intestine. Mice were maintained on a diet containing soybean oil, with or without PA, for 2 mo. (A) Mononuclear cells were isolated from the small and large intestines for the ﬂow cytometric analysis of IgA+ B2202 PCs. Data are representative of three independent experiments. (B) Mice were orally immunized with OVA plus CT on days 0, 7, and 14. On day 21, mononuclear cells were isolated from the small and large intestine for the detection of OVA-speciﬁc IgA Ab–forming cells by ELISPOT assay. Data are mean 6 1SD(n =6/ group from two separate experiments). (C) The metabolic pathway mediated by SPT to generate sphingolipids from PA. (D) The frequency of IgA+ B2202 PCs in the large intestine of mice maintained on a PA-enriched diet and myriocin. The graphs show data from individual samples, and the horizontal lines indicate the means. Similar results were obtained from two independent experiments. 1670 DIETARY PALMITIC ACID IN INTESTINAL IgA PRODUCTION

The increased proliferation of IgA PCs in the large intestine of IgA production from PCs in a TLR4-independent manner in our mice maintained with a PA-enriched diet current study. In line with this finding, several groups reported that We performed BrdU-uptake assays to examine the effects of PA on TLR2 acts as a receptor for PA or that PA enters cells where it the proliferation and survival of IgA PCs in the large intestine. Soon induces signal transduction for the consequent production of in- after BrdU administration (day 1), the proportions of BrdU+ and flammatory cytokines (33–36). BrdU2 IgA+ cells in the large intestine were higher when mice In addition to PA’s direct effect on IgA-producing PCs, the SPT- were maintained on a PA-enriched diet than when they were mediated metabolism of PA is involved in the PA-mediated en- maintained on the control diet, but the increase ratio of IgA+ cells hancement of intestinal IgA responses. SPT is an essential enzyme was higher in BrdU+ IgA+ cells than in BrdU2 IgA+ cells for the de novo pathway of sphingolipid synthesis, in which (Fig. 7A). These findings suggest that PA metabolites induced the palmitoyl-CoA and serine act as substrates of SPT to generate 3- proliferation of IgA+ cells in the large intestine. In contrast, on day keto-dihydosphingosine, with subsequent conversion into other 4, both the control and PA-enriched groups showed similar levels sphingolipids, such as sphingomyelin, ceramide, sphingosine, and of BrdU+ IgA+ cells (Fig. 7B). These data collectively suggested S1P (37, 38). Sphingolipids are a class of membrane lipids that that PA metabolites primarily induced the proliferation of IgA+ also are known to have biologic functions (27, 28). For example, cells in the large intestine rather than prolonged their survival. ceramide regulates cytoskeletal changes, cell cycle, and apoptosis (27, 28). Accordingly, perhaps an increase in ceramide concen- Discussion trations induced the proliferation or prolonged the survival of IgA PCs, subsequently increasing the number of IgA-producing PCs in In the current study, we extended our knowledge of lipid-mediated the large intestine. In addition, in the extracellular compartment, Downloaded from immune regulation by showing the immunologic function of di- S1P controls cell trafficking by recruiting cells toward regions etary PA in the enhancement of intestinal IgA responses. Unlike with high concentrations of S1P (39). Of note, we previously re- the sterile environment of systemic immune compartments (e.g., ported that S1P regulates intestinal IgA responses by controlling spleen), intestinal tissues are continuously exposed to exogenous trafficking of IgA+ cells from inductive sites (e.g., PPs and peri- factors, including commensal bacteria and dietary materials and toneal cavity) into the iLP (22, 40). Therefore, another possibility actively use them to establish a homeostatic inflammatory condition

is that the PA-enriched diet induced an increase in the intestinal http://www.jimmunol.org/ (6). Indeed, in contrast to the massive inﬂammatory responses in- extracellular S1P concentration, resulting in the effective recruit- duced by the systemic injection of bacterial products, such as LPS ment of IgA PCs into the intestine. Our current ﬁndings suggest (e.g., sepsis), the intestinal immune system requires bacterial stimu- that at least one of the enhancing effects of the PA-enriched diet lation for its maturation (29). Our current study demonstrates that was mediated by the induction of proliferating IgA+ PCs in the dietary PA can augment intestinal IgA responses when an ap- large intestine. propriate amount of dietary oil (4%) is supplied. This effect PA was not only included in the diet but was also generated contrasts sharply with the deleterious plasma PA levels that are through de novo lipogenesis, whereby carbohydrates are converted induced by a high-fat diet and are considered to be a risk factor for to PA. The de novo pathway achieves stable concentrations of PA

inflammation and diabetes (30). Therefore, like bacterial products, by guest on September 29, 2021 in vivo. This pathway can explain the specificity of the effect of PA has the opposite immunologic effect on intestinal and systemic dietary PA enrichment on the PA content in various tissues. In this immune compartments and actually plays a beneficial role in the study, we found that the increases in PA in the PA-enriched diet maturation of the intestinal immune system. group were specific to the intestinal tissues and not the serum. This The enhancement of intestinal IgA responses by dietary PA is tissue specificity is consistent with the specific effect of dietary PA, mediated by at least two distinct pathways. One is PA’s direct effect which increased intestinal IgA responses without affecting serum on IgA-producing PCs, and the other is mediated by PA-derived Ab production. Mice fed high-fat diets show increased serum PA metabolites, sphingolipids. In addition to these two pathways, PA levels, which are a risk factor for inflammation and diabetes (30). affects APCs (e.g., macrophages and dendritic cells) to promote However, our current findings indicate that the increased propor- Ag presentation and the production of cytokines, including IL-6 tion of PA in the dietary oil did not affect serum PA levels when and TNF-a; this effect is at least partly mediated by TLR4 (31, 32) the overall amount of oil consumed was normal (4%). and represents a plausible third pathway to enhancing intestinal Intestinal tissues contain higher levels of sphingolipids than do IgA production. Unlike the effect of PA on APCs, PA promoted other tissues (41), and we found that the effect of an SPT inhibitor was selective for the large, but not small, intestine. One of the major differences between the small and large intestines is the amount of commensal bacteria. We recently reported that some lipid metabolic pathways are uniquely mediated by commensal bacteria (42), raising the possibility that commensal bacteria may affect sphingolipid metabolism. However, germ-free and specific pathogen–free rats have comparable levels of sphingolipids in the intestine (43). Therefore, it is plausible that commensal bacteria are unlikely to participate in this pathway. In contrast, the expression pattern of enzymes involved in the generation of sphingolipids differs between intestinal compartments (44, 45), indicating that the differences in sphingolipid metabolism between the small and large FIGURE 7. A PA-enriched diet induces the proliferation of IgA PCs. intestines determine their differing dependence on SPT in PA- Mice were maintained on a diet containing soybean oil, with or without PA, for 2 mo, after which BrdU was injected i.p. (day 0). On day 1 (A) and mediated intestinal IgA responses. day 4 (B), mononuclear cells isolated from the large intestine were ana- Taken together, our current findings demonstrate that dietary PA lyzed by flow cytometry to determine the proportion of BrdU+ IgA+ PCs. and its metabolites play a critical role in the enhancement of in- Data are mean 6 1SD(n = 4). Similar results were obtained from two testinal IgA responses. This information can be applied to the independent experiments. development of mucosal adjuvants. The Journal of Immunology 1671

Disclosures 24. Maloney, E., I. R. Sweet, D. M. Hockenbery, M. Pham, N. O. Rizzo, S. Tateya, P. Handa, M. W. Schwartz, and F. Kim. 2009. Activation of NF-kappaB by The authors have no financial conflicts of interest. palmitate in endothelial cells: a key role for NADPH oxidase-derived superoxide in response to TLR4 activation. Arterioscler. Thromb. Vasc. Biol. 29: 1370–1375. 25. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. Van Huffel, X. Du, D. Birdwell, References E. Alejos, M. Silva, C. Galanos, et al. 1998. Defective LPS signaling in C3H/HeJ 1. Macpherson, A. J., K. D. McCoy, F. E. Johansen, and P. Brandtzaeg. 2008. The and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085–2088. immune geography of IgA induction and function. Mucosal Immunol. 1: 11–22. 26. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda, 2. Woof, J. M., and M. A. Kerr. 2006. The function of immunoglobulin A in im- and S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are munity. J. Pathol. 208: 270–282. hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene 3. Kunisawa, J., Y. Kurashima, and H. Kiyono. 2012. Gut-associated lymphoid product. J. Immunol. 162: 3749–3752. tissues for the development of oral vaccines. Adv. Drug Deliv. Rev. 64: 523–530. 27. Fyrst, H., and J. D. Saba. 2010. An update on sphingosine-1-phosphate and other 4. Fagarasan, S., S. Kawamoto, O. Kanagawa, and K. Suzuki. 2010. Adaptive sphingolipid mediators. Nat. Chem. Biol. 6: 489–497. immune regulation in the gut: T cell-dependent and T cell-independent IgA 28. Hannun, Y. A., and L. M. Obeid. 2008. Principles of bioactive lipid signalling: synthesis. Annu. Rev. Immunol. 28: 243–273. lessons from sphingolipids. Nat. Rev. Mol. Cell Biol. 9: 139–150. 5. Cerutti, A., K. Chen, and A. Chorny. 2011. Immunoglobulin responses at the 29. Kayama, H., and K. Takeda. 2012. Regulation of intestinal homeostasis by innate mucosal interface. Annu. Rev. Immunol. 29: 273–293. and adaptive immunity. Int. Immunol. 24: 673–680. 6. Spencer, S. P., and Y. Belkaid. 2012. Dietary and commensal derived nutrients: 30. Kennedy, A., K. Martinez, C. C. Chuang, K. LaPoint, and M. McIntosh. 2009. shaping mucosal and systemic immunity. Curr. Opin. Immunol. 24: 379–384. Saturated fatty acid-mediated inflammation and insulin resistance in adipose 7. Weinstein, P. D., and J. J. Cebra. 1991. The preference for switching to IgA tissue: mechanisms of action and implications. J. Nutr. 139: 1–4. expression by Peyer’s patch germinal center B cells is likely due to the intrinsic 31. Weigert, C., K. Brodbeck, H. Staiger, C. Kausch, F. Machicao, H. U. Ha¨ring, and influence of their microenvironment. J. Immunol. 147: 4126–4135. E. D. Schleicher. 2004. Palmitate, but not unsaturated fatty acids, induces the 8. Hamada, H., T. Hiroi, Y. Nishiyama, H. Takahashi, Y. Masunaga, S. Hachimura, expression of interleukin-6 in human myotubes through proteasome-dependent S. Kaminogawa, H. Takahashi-Iwanaga, T. Iwanaga, H. Kiyono, et al. 2002. activation of nuclear factor-kappaB. J. Biol. Chem. 279: 23942–23952. 32. Weatherill, A. R., J. Y. Lee, L. Zhao, D. G. Lemay, H. S. Youn, and

Identification of multiple isolated lymphoid follicles on the antimesenteric wall Downloaded from of the mouse small intestine. J. Immunol. 168: 57–64. D. H. Hwang. 2005. Saturated and polyunsaturated fatty acids reciprocally 9. Kunisawa, J., M. Gohda, E. Hashimoto, I. Ishikawa, M. Higuchi, Y. Suzuki, modulate dendritic cell functions mediated through TLR4. J. Immunol. 174: Y. Goto, C. Panea, and I.I. Ivanov, R. Sumiya, et al. 2013. Microbe-dependent 5390–5397. CD11b+ IgA+ plasma cells mediate robust early-phase intestinal IgA responses 33. Snodgrass, R. G., S. Huang, I. W. Choi, J. C. Rutledge, and D. H. Hwang. 2013. b in mice. Nat. Commun. 4: 1772. Inflammasome-mediated secretion of IL-1 in human monocytes through TLR2 10. Maslowski, K. M., and C. R. Mackay. 2011. Diet, gut microbiota and immune activation; modulation by dietary fatty acids. J. Immunol. 191: 4337–4347. responses. Nat. Immunol. 12: 5–9. 34. Zhao, L., M. J. Kwon, S. Huang, J. Y. Lee, K. Fukase, N. Inohara, and 11. Kunisawa, J., and H. Kiyono. 2013. Vitamin-mediated regulation of intestinal D. H. Hwang. 2007. Differential modulation of Nods signaling pathways by fatty immunity. Front. Immunol. 4: 189. acids in human colonic epithelial HCT116 cells. J. Biol. Chem. 282: 11618– http://www.jimmunol.org/ 12. Hanson, L. A., A.-K. Robertson, J. Bjersing, and M. V. Herias. 2005. Under- 11628. nutrition, immunodeficiency, and mucosal infections. In Mucosal Immunology, 35. Erridge, C., and N. J. Samani. 2009. Saturated fatty acids do not directly stim- 3rd ed. J. Mestecky, M. E. Lamm, W. Strober, J. Bienenstock, J. R. McGhee, and ulate Toll-like receptor signaling. Arterioscler. Thromb. Vasc. Biol. 29: 1944– L. Mayer, eds. Academic Press, San Diego, CA, p. 1159–1178. 1949. 13. Wintergerst, E. S., S. Maggini, and D. H. Hornig. 2007. Contribution of selected 36. Schwartz, E. A., W. Y. Zhang, S. K. Karnik, S. Borwege, V. R. Anand, vitamins and trace elements to immune function. Ann. Nutr. Metab. 51: 301–323. P. S. Laine, Y. Su, and P. D. Reaven. 2010. Nutrient modification of the innate 14. Galli, C., and P. C. Calder. 2009. Effects of fat and fatty acid intake on in- immune response: a novel mechanism by which saturated fatty acids greatly flammatory and immune responses: a critical review. Ann. Nutr. Metab. 55: 123– amplify monocyte inflammation. Arterioscler. Thromb. Vasc. Biol. 30: 802–808. 139. 37. Merrill, A. H., Jr. 1983. Characterization of serine palmitoyltransferase activity 15. Margioris, A. N. 2009. Fatty acids and postprandial inflammation. Curr. Opin. in Chinese hamster ovary cells. Biochim. Biophys. Acta 754: 284–291. Clin. Nutr. Metab. Care 12: 129–137. 38. Gault, C. R., L. M. Obeid, and Y. A. Hannun. 2010. An overview of sphingolipid 16. Arita, M. 2012. Mediator lipidomics in acute inflammation and resolution. J. metabolism: from synthesis to breakdown. Adv. Exp. Med. Biol. 688: 1–23. by guest on September 29, 2021 Biochem. 152: 313–319. 39. Cyster, J. G., and S. R. Schwab. 2012. Sphingosine-1-phosphate and lymphocyte 17. Jin, C., and R. A. Flavell. 2013. Innate sensors of pathogen and stress: linking egress from lymphoid organs. Annu. Rev. Immunol. 30: 69–94. inflammation to obesity. J. Allergy Clin. Immunol. 132: 287–294. 40. Kunisawa, J., Y. Kurashima, M. Gohda, M. Higuchi, I. Ishikawa, F. Miura, 18. Schmitz, G., and J. Ecker. 2008. The opposing effects of n-3 and n-6 fatty acids. I. Ogahara, and H. Kiyono. 2007. Sphingosine 1-phosphate regulates peritoneal Prog. Lipid Res. 47: 147–155. B-cell trafficking for subsequent intestinal IgA production. Blood 109: 3749– 19. Nguyen, M. T., S. Favelyukis, A. K. Nguyen, D. Reichart, P. A. Scott, A. Jenn, 3756. R. Liu-Bryan, C. K. Glass, J. G. Neels, and J. M. Olefsky. 2007. A subpopulation 41. Bouhours, D., and J. F. Bouhours. 1985. Developmental changes of mono- of macrophages infiltrates hypertrophic adipose tissue and is activated by free hexosylceramide and free ceramide in the large intestine of the rat. J. Biochem. fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways. J. Biol. 98: 1359–1366. Chem. 282: 35279–35292. 42. Gustafsson, B. E., K. A. Karlsson, G. Larson, T. Midtvedt, N. Stro¨mberg, 20. Ha˚versen, L., K. N. Danielsson, L. Fogelstrand, and O. Wiklund. 2009. Induction S. Teneberg, and J. Thurin. 1986. Glycosphingolipid patterns of the gastroin- of proinflammatory cytokines by long-chain saturated fatty acids in human testinal tract and feces of germ-free and conventional rats. J. Biol. Chem. 261: macrophages. Atherosclerosis 202: 382–393. 15294–15300. 21. He, Q., R. T. Riley, and R. P. Sharma. 2005. Myriocin prevents fumonisin B1- 43. Kishino, S., M. Takeuchi, S. B. Park, A. Hirata, N. Kitamura, J. Kunisawa, induced sphingoid base accumulation in mice liver without ameliorating hepa- H. Kiyono, R. Iwamoto, Y. Isobe, M. Arita, et al. 2013. Polyunsaturated fatty totoxicity. Food Chem. Toxicol. 43: 969–979. acid saturation by gut lactic acid bacteria affecting host lipid composition. Proc. 22. Gohda, M., J. Kunisawa, F. Miura, Y. Kagiyama, Y. Kurashima, M. Higuchi, Natl. Acad. Sci. USA 110: 17808–17813. I. Ishikawa, I. Ogahara, and H. Kiyono. 2008. Sphingosine 1-phosphate regulates 44. Duan, R. D., L. Nyberg, and A. Nilsson. 1995. Alkaline sphingomyelinase ac- the egress of IgA plasmablasts from Peyer’s patches for intestinal IgA responses. tivity in rat gastrointestinal tract: distribution and characteristics. Biochim. J. Immunol. 180: 5335–5343. Biophys. Acta 1259: 49–55. 23. Schaeffler, A., P. Gross, R. Buettner, C. Bollheimer, C. Buechler, M. Neumeier, 45. Kono, M., J. L. Dreier, J. M. Ellis, M. L. Allende, D. N. Kalkofen, A. Kopp, J. Schoelmerich, and W. Falk. 2009. Fatty acid-induced induction of K. M. Sanders, J. Bielawski, A. Bielawska, Y. A. Hannun, and R. L. Proia. 2006. Toll-like receptor-4/nuclear factor-kappaB pathway in adipocytes links nutri- Neutral ceramidase encoded by the Asah2 gene is essential for the intestinal tional signalling with innate immunity. Immunology 126: 233–245. degradation of sphingolipids. J. Biol. Chem. 281: 7324–7331.