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Article Cite This: J. Agric. Food Chem. 2018, 66, 108−117

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Short-Chain Fatty Acids Suppress Inflammatory Reactions in Caco‑2 Cells and Mouse Colons Tran Van Hung†,‡ and Takuya Suzuki*,† †

Department of Biofunctional Science and Technology, Graduate School of Biosphere Science, Hiroshima University, Higashihiroshima 739-8528, Japan ‡ Ho Chi Minh City University of Food Industry, Ho Chi Min 15, Vietnam S Supporting Information *

ABSTRACT: Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, play an important role in the maintenance of intestinal homeostasis. In the present study, anti-inflammatory effects of SCFAs were examined in human intestinal Caco-2 cells and mouse colonic cultures. Stimulation of Caco-2 cells with tumor necrosis factor (TNF)-α induced interleukin (IL)-8 (TNF-α, 17.1 ± 7.2 vs Control, 1.00 ± 0.26, P < 0.01) and IL-6 expression (TNF-α, 21.7 ± 10.0 vs Control, 1.00 ± 0.28, P < 0.01) through the activation of nuclear factor κB p65, spleen tyrosine kinase, and mitogen-activated protein kinase pathways. Pretreatment of cells with acetate (5 mM, IL-8 1.23 ± 0.40, IL-6 2.19 ± 0.92, P < 0.01 ), propionate (2.5 mM, IL-8 2.45 ± 2.10, IL-6 2.19 ± 0.92, P < 0.01), or butyrate (0.625 mM, IL-8 1.44 ± 0.70, IL-6 2.31 ± 0.32, P < 0.01) suppressed inflammatory responses induced by TNF-α. Pharmacological inhibition of monocarboxylate transporter (MCT)-1 attenuated the suppression of inflammatory signals by SCFAs. High expression levels of CXC motif chemokine ligand 2 (CXCL2, an IL-8 homologue, DSS, 31.7 ± 9.8 vs Control, 1.00 ± 0.70, P < 0.01) and IL-6 (DSS, 17.5 ± 7.2 vs Control, 1.00 ± 0.68, P < 0.01) were observed in BALB/c mouse colonic cultures exposed to dextran sodium sulfate, whereas treatments with mixtures of SCFAs decreased these elevated expression levels (CXCL2 4.14 ± 2.88, IL-6 0.58 ± 0.28, P < 0.01). Our results suggest that SCFAs transported by MCT-1 suppress TNF-α-induced inflammatory signaling in intestinal cells. KEYWORDS: interleukin-8, intestinal epithelial cells, monocarboxylate transporter-1, short-chain fatty acids, tumor necrosis factor-α

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INTRODUCTION The intestinal microbiota is essential for fermentation of undigested carbohydrates and proteins. Various bacterial metabolites produced as a result of fermentation exert intestinal and systemic regulatory effects. One major group of these bacterial metabolites with pleiotropic effects comprises short-chain fatty acids (SCFAs). SCFAs are the major end products of bacterial fermentation in the large intestine, with acetate (2-carbon), propionate (3-carbon), and butyrate (4carbon) being the predominant metabolites.1,2 SCFAs, especially butyrate, serve as the major energy source for colonic epithelial cells and have been also shown to influence different cellular processes and functions in the colon, such as cell differentiation, cell apoptosis, colonic motility, and electrolyte absorption.3−5 Recent studies demonstrated that SCFAs, especially butyrate, also regulate intestinal inflammation.6−8 Butyrate induced differentiation of colonic regulatory T cells and reduced experimental colitis in mice through histone deacetylase inhibition.9 The inflammatory responses of intestinal epithelial cells to bacteria and their components are attenuated by butyrate. 10−13 Our previous study also demonstrated that consumption of highly fermentable dietary fiber that generates large amounts SCFAs in the colons ameliorated intestinal inflammation in mice with induced colitis.14 However, the precise mechanisms underlying SCFAmediated regulation of intestinal inflammation are still not fully understood. SCFAs regulate several cellular processes through apparently different mechanisms.15,16 Recent studies have demonstrated © 2017 American Chemical Society

that SCFAs activate at least four different G protein-coupled receptors (GPCRs): GPR43, GPR41, GPR109a, and olfactory receptor 78,17,18 which are reportedly expressed in intestinal epithelial cells. It is believed that colonic mucosa absorbs SCFAs via simple diffusion of their nonionic forms and via SCFA−/HCO3− exchange.19,20 However, with the acidic dissociation constant of ∼4.8, the majority of SCFAs are expected to be ionized at physiological pH in colonic lumen. In this regard, a number of studies have reported evidence for the presence of carrier-mediated pathways for the absorption of ionized SCFAs. SCFAs have been shown to be substrates of monocarboxylate transporters (MCTs), either H+ cotransporters or HCO3− antiporters.21 Previous studies using pharmacological inhibition, analog competition, and antisense techniques indicated that MCT-1 is involved in luminal SCFA uptake by intestinal epithelial cells.22−25 In addition, immunohistochemical and immunoblot analyses of human biopsies revealed that MCT-1 is expressed and localized on the apical membrane of colonocytes.26 MCT-1 expression and localization in intestinal cells is influenced by different stimulations.27−30 However, the extent of MCT-1 involvement in the inflammatory regulation of SCFAs in colons still remains to be elucidated.31 Received: Revised: Accepted: Published: 108

September 11, 2017 December 12, 2017 December 13, 2017 December 13, 2017 DOI: 10.1021/acs.jafc.7b04233 J. Agric. Food Chem. 2018, 66, 108−117

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Journal of Agricultural and Food Chemistry

the basolateral and apical sides of Caco-2 cells, respectively. SCFA concentrations used were slightly lower than those found in human feces,37,38 because Caco-2 and T84 cells do not possess the mucous layer that provides a physical barrier to luminal solutes, including SCFAs, in the colon. To examine the specificity of the three SCFAs, valerate, lactate, and succinate at a concentration of 0.5 mM were also used. To eliminate the effects of different osmolarity on the results, isotonic test solutions (300 mOsmol/L) containing 5× concentrated SCFAs were prepared with sodium chloride and 0.2 mL of the 5× concentrates were mixed with 0.8 mL of the regular cell culture medium to achieve the required final solution composition. For example, a mixture of 100 mM sodium acetate and 50 mM NaCl, indicating osmolarity of 300 mOsmol/L, was used for the treatment with 20 mM acetate. Other test solutions were prepared in the same manner. A 150 mM NaCl solution was used for control treatment. Because we used sodium salts of these acids, substantial changes in pH of the final solutions were not observed. In some experiments, a simultaneous treatment with α-CHA (a MCT-1 inhibitor) was performed. Culture media were collected 24 h after TNF-α administration to determine IL-8 concentrations by enzyme-linked immune sorbent assay (ELISA) as described below. Caco-2 cell extracts were collected for qRT-PCR and immunoblot analyses at 0.5 and 24 h as described below. In addition, the viability of Caco-2 cells incubated with or without acetate (0−20 mM), propionate (0−10 mM), or butyrate (0−2.5 mM) in the presence or absence of TNF-α was examined using a commercially available kit (Cell Counting Kit-8; Dojindo Laboratories). Animals. All study protocols were approved in advance by the Animal Use Committee of the Hiroshima University, and all mice were maintained in accordance with the guidelines for the care and use of laboratory animals of the Hiroshima University (permission number: C15−10−2). Male BALB/c mice, aged 7 weeks and weighing about 21 g, were obtained from Charles River, Inc. (Yokohama, Japan) and housed under standard laboratory conditions: temperature 22 ± 2 °C, relative humidity 40−60%, and lights on during 08:00−20:00 h throughout the study. The mice were allowed to acclimatize to the laboratory environment with free access to AIN93G control diet and distilled water for 1 week before the start of the experiment. Experimental Design of the Animal Study. Mice (n = 12) were randomly allocated to two groups: control and DSS. Both groups were provided with control diet throughout the study. DSS group received 2% (w/v) DSS in drinking water for 6 days, followed by distilled water for 4 days, whereas control mice received distilled water only. Body weight of mice and clinical score for colitis were evaluated every day after the start of DSS treatment as described previously.39 The clinical score was calculated as the sum of scores for diarrhea, bloody stools, and weight loss (Table S1). At the end of the experiment (day 10), the mice were sacrificed by exsanguination under isoflurane anesthesia, and the colon tissues were dissected and subjected to hematoxylin and eosin (H&E) staining, myeloperoxidase (MPO) assay, and SCFA treatment, as described below. Treatment of Colon Tissues. Colon tissues (2 cm long distal portions) removed from mice were rinsed with ice-cold Hanks’ balanced salt solution and cut into four 5 mm long pieces. Specimens from each mouse were allocated to four treatment groups: control, LSCFAs, M-SCFAs, and H-SCFAs. Specimens were incubated with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 100 mL/L fetal bovine serum with or without SCFAs (acetatepropionate-butyrate 20:10:2.5 for H-SCFAs, 10:5:1.25 for M-SCFAs, and 5:2.5:0.625 for L-SCFAs, where numbers indicate concentrations in mM) in 24-well plates. Concentrations of SCFAs and compositions of SCFA-containing solutions were similar to those used in our cell culture studies. Control specimens were incubated with DMEM without SCFAs. Colon specimens were collected after a 24-h incubation to determine CXCL-2 (mouse homologue for IL-8), IL6, and IL-10 mRNA expression levels by qRT-PCR. Measurement of IL-8 Production. IL-8 concentrations in cell culture media were determined by ELISA (Human CXCL-8/IL-8, DuoSet ELISA, R&D systems, Minneapolis, MN, USA) according to

Intestinal inflammation develops through different cellular processes. A hallmark of intestinal inflammation is the recruitment of neutrophils from blood to inflammatory sites. Accumulating evidence indicates that interleukin (IL)-8 secreted by intestinal epithelial cells has an important role in neutrophil recruitment. Previous studies showed that neutralizing antibodies against murine IL-8 homologues reduced neutrophil infiltration into the mucosa and alleviated clinical symptoms in mice with experimentally induced colitis.32 In the mucosa of patients with inflammatory bowel diseases (IBDs), such as ulcerative colitis (UC) and Crohn’s disease (CD), the infiltration of neutrophils with higher levels of IL-8 expression correlated with disease severity.33 IL-8 secretion is low in intact epithelium, but a wide range of stimuli, e.g., pro-inflammatory tumor necrosis factor (TNF)-α, rapidly and potently induce IL-8 production. Thus, suppression of TNF-α-induced increase in IL-8 expression could be an effective approach to prevent intestinal inflammation and to treat IBDs.33 In addition to TNF-α and IL-8, other cytokines, such as IL-6 and IL-10, seem to be closely involved in the regulation of intestinal inflammation. Expression of IL-6 as well as of IL-8 in intestinal epithelial cells is induced by TNF-α. IL-6, in turn, promotes the development of helper T17 cells and reduces intestinal barrier integrity.34 Anti-inflammatory IL-10, which is produced largely by regulatory T cells, suppresses the excess immunological response, as shown by the fact that IL-10-deficient mice develop colitis spontaneously.35 The aims of the present study were to investigate antiinflammatory regulation by SCFAs and to identify molecular mechanisms underlying this process using human intestinal Caco-2 cells and cultured mouse colons. We focused particularly on IL-8 production and cellular mechanisms of its regulation.

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MATERIALS AND METHODS

Chemicals. Cell culture reagents and supplies were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Rabbit polyclonal anti-pp38 MAPK (Thr-180/Tyr-182) and rabbit monoclonal antibodies against pJNK1/2 (Thr-183/Tyr-185), pERK1/2 (Thr-202/Tyr-204), pSyk (Tyr-525/Tyr-526), and pNF-κB p65 (Ser536) were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated antirabbit IgG and antimouse IgG were purchased from SeraCare (Milford, MA, USA). The MCT-1 inhibitor α-cyano-4-hydroxycinnamic acid (α-CHA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dextran sodium sulfate (DSS, molecular weight 36 000−50 000) was purchased from MP Biomedicals (Santa Ana, CA, USA). All other chemicals were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cell Culture. Human intestinal Caco-2 and T84 cells (HTB-37 and CCL-248; American Type Culture Collection, Manassas, VA, USA) were propagated and maintained under standard cell culture conditions as described previously.36 The cells were grown in 24-well plates (Corning Incorporated, Kennebunk, ME, USA) and permeable polyester membrane filter supports (Transwell, 12 mm diameter, 0.4 μm pore size; Corning Costar Co.). All experiments were conducted on days 13−14 in Caco-2 cells and 18−21 in T84 cells postseeding, when cells reached confluence and became differentiated. Cultures were used between passages 48 and 65 in Caco-2 cells and 68 and 75 in T84 cells, and the medium was refreshed every 3 days. Treatment of Intestinal Caco-2 and T84 Cells. Caco-2 and T84 cell monolayers were incubated without or with acetate (0−20 mM), propionate (0−10 mM), or butyrate (0−2.5 mM) in the absence or presence of 30 ng/mL TNF-α. TNF-α was added to cell culture medium 1 h after the administration of SCFAs. In the experiment using Transwell system, TNF-α and SCFA were added to 109

DOI: 10.1021/acs.jafc.7b04233 J. Agric. Food Chem. 2018, 66, 108−117

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Figure 1. Sensitivity of TNF-α-induced increase in IL-8 and IL-6 expression levels in Caco-2 cells to short-chain fatty acids (SCFAs). Caco-2 cells were incubated for 24 h in the absence or presence of 30 ng/mL TNF-α. Acetate (A, 5−20 mM, B and C, 5 mM), propionate (A, 2.5−10 mM, B and C, 2.5 mM), or butyrate (A, 0.625−2.5 mM, B and C, 0.625 mM) were added to cell cultures 1 h before TNF-α. IL-8 concentration in cell culture supernatant was measured by enzyme-linked immune sorbent assay (A). Expression levels of IL-8 (B) and IL-6 (C) mRNAs were determined by qRT-PCR. Data are represented as the mean ± SEM of three independent experiments. Means without a common letter differ at the following level of statistical significance: P < 0.05. the manufacturer’s protocol. Absorbance at 450 nm was measured using a microplate spectrophotometer (Multiskan Go, Thermo Fisher Scientific Inc.). qRT-PCR Analysis. Total RNA from Caco-2 cells and mouse colon tissues was isolated using TRI reagent (Sigma-Aldrich) and NucleoSpin RNA II (Macherey-Nagel, Bethlehem, PA, USA), respectively. RNA was reverse-transcribed into cDNA using a ReverTra Ace qPCR RT kit (TOYOBO, Osaka, Japan), according to the manufacturer’s protocol. The primer sequences used for PCR are shown in Table S2. qRT-PCR was performed using Step One Real-Time PCR system (Life Technologies, Waltham, MA, USA) and KAPA SYBR FAST qPCR kit (KAPA Biosystems, Wilmington, MA, USA). Data were analyzed by the 2−ΔΔCt method and normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), and hypoxanthine-guanine phosphoribosyltransferase (HPRT)-1 genes used as internal controls, as described previously.14,40 Immunoblot Analysis. Protein extracts from Caco-2 cells were mixed with a half volume of Laemmli sample buffer (3×) as described previously.14,36 Proteins (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gel and transferred to polyvinylidene difluoride membranes (Immobilon-P, Merck Millipore, Billerica, MA, USA). Membranes were blotted for pNFκB p65, pERK1/2, pSyk, pp38 MAPK, and pJNK1/2 using specific antibodies in combination with HRP-conjugated antimouse IgG or antirabbit IgG antibodies. The blots were developed using enhanced

chemiluminescence detection reagents (PerkinElmer Life Sciences, Waltham, MA, USA). Quantification was performed by densitometric analysis of specific bands on the immunoblots using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Hematoxylin and Eosin Staining. Mouse colon tissues were embedded in OCT compound (Sakura Finetek USA Inc., CA, USA) and frozen 8-μm thick sections were prepared on glass slides and subjected to H&E staining. The specimens were preserved in Eukitt mounting medium, and images were collected using Nikon Eclipse E600 (Tokyo, Japan). MPO Assay. MPO activity in colonic tissues was determined by a standard enzymatic procedure as described previously.14 Statistical Analysis. All data are presented as the mean ± standard error of the mean (SEM). Expression levels of the IL6 and IL8 genes in Caco-2 cells and of CXCL2, IL6, and IL10 genes in mouse colon were normalized to expression levels of three housekeeping genes, GAPDH, ACTB, and HPRT1. Data normalized to GAPDH expression levels are shown as representatives, because similar results were obtained by normalizing to the levels of the other two housekeeping genes. Statistical analyses were performed using one-way ANOVA followed by the Tukey-Kramer post hoc test. All statistical analyses were conducted with a significance level of 0.05 using the Statistical Package for the Social Sciences (SPSS) version 18 for Windows (SPSS Inc., Chicago, IL, USA). 110

DOI: 10.1021/acs.jafc.7b04233 J. Agric. Food Chem. 2018, 66, 108−117

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Figure 2. Structural specificity of SCFA-mediated IL-8 secretion by Caco-2 cells. Caco-2 cells were incubated for 24 h in the absence or presence of 30 ng/mL TNF-α. Acetate, propionate, butyrate, valerate, lactate, or succinate at a concentration of 0.5 mM were added to cell cultures 1 h before TNF-α treatment. IL-8 concentration in cell culture supernatants was measured by the enzyme-linked immune sorbent assay. Data are represented as the mean ± SEM of three independent experiments. Means without a common letter differ at the following level of statistical significance: P < 0.05.

Figure 3. Reduction of TNF-α-induced enhancement of cellular signaling in Caco-2 cells by SCFAs. Caco-2 cells were incubated for 30 min in the absence or presence of 30 ng/mL TNF-α. Acetate (5 mM), propionate (2.5 mM), or butyrate (0.625 mM) were added to cell cultures 1 h before TNF-α. Levels of phosphorylated p65 NF-κB, Syk, ERK1/2, JNK1/2, and p38 MAPK were analyzed by immunoblotting. Data are represented as the mean ± SEM of three independent experiments. Means without a common letter differ at the following level of statistical significance: P < 0.05.

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RESULTS

to apical and basolateral sides, respectively. In such conditions, there was no interaction between SCFAs and TNF-α, whereas the decrease in IL-8 levels caused by SCFAs persisted (Figure S1). In addition, SCFAs suppressed TNF-α-induced IL-8 production in another intestinal cell line, T84, indicating that these effects were not cell line-specific (Figure S2). TNF-α increased expression of IL-8 and IL-6 mRNAs at 24 h (Figure 1B, P < 0.05), and, as in the above-mentioned experiments, these increases were suppressed by 5 mM acetate, 2.5 mM propionate, or 0.625 mM butyrate. The viability of cells, as assessed by a cytotoxicity assay, was not influenced by TNF-α or SCFAs (data not shown). To examine the specificity of the three selected SCFAs, three other carboxylic acids, valerate, lactate, and succinate, were used at a concentration of 0.5 mM (Figure 2). IL-8 secretion by Caco-2 cells stimulated by TNF-α was suppressed by succinate as well as by acetate, propionate, and butyrate. In contrast, valerate, which has a longer hydrocarbon chain, and lactate, a hydroxy carboxylic acid, did not reduce TNF-αinduced IL-8 secretion.

Reduction of TNF-α-Induced Increase in IL-8 and IL-6 Expression Levels in Caco-2 Cells by SCFAs. To examine anti-inflammatory effects of SCFAs in intestinal epithelium, Caco-2 cells, which are known to be a valid model of human intestinal epithelial cells, were stimulated by TNF-α and incubated with acetate, propionate, or butyrate at different concentrations. Secretion of IL-8 by Caco-2 cells was potently increased by TNF-α in control cells, whereas concomitant treatment of cells with acetate, propionate, or butyrate at all concentrations tested reduced this enhancement of IL-8 secretion (Figure 1A, P < 0.01). Because these SCFAs tended to be more effective at lower concentrations, in the remaining experiments, the concentrations of acetate, propionate, and butyrate were set to 5, 2.5, and 0.625 mM, respectively. To eliminate the possibility that SCFAs chemically interacted with TNF-α, thereby resulting in the reduction of IL-8 production, Caco-2 cells grown on permeable filter supports were used. In this system, SCFAs and TNF-α were administered separately 111

DOI: 10.1021/acs.jafc.7b04233 J. Agric. Food Chem. 2018, 66, 108−117

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Figure 4. SCFA-mediated reduction of IL-8 expression induced by TNF-α was sensitive to pharmacological inhibition of MCT-1 in Caco-2 cells. Caco-2 cells were incubated for 24 h (A) or 30 min (B) in the absence or presence of 30 ng/mL TNF-α. Acetate (5 mM), propionate (2.5 mM), or butyrate (0.625 mM) were added to cell cultures 1 h before TNF-α. The MCT-1 inhibitor α-CHA (10 mM) was added to cell cultures 1 h before administration of SCFAs. (A) IL-8 concentration in cell culture supernatant was measured by the enzyme-linked immunosorbent assay. (B) Levels of phosphorylated Syk, ERK1/2, and p38 MAPK were analyzed by immunoblotting. Data are represented as the mean ± SEM of three independent experiments. Means without a common letter differ at the following level of statistical significance: P < 0.05.

Pretreatment of Caco-2 cells with α-CHA, an MCT-1 inhibitor, for 1 h completely inhibited the suppression of TNF-α-induced IL-8 secretion by acetate, propionate, or butyrate (Figure 4A, P < 0.01). Similarly, the MCT-1 inhibitor reduced suppressive effects of acetate, propionate, and butyrate on TNF-α-induced phosphorylation of Syk, ERK1/2, and p38 MAPK at 30 min (Figure 4B, P < 0.01). α-CHA treatment alone did not influence IL-8 production in the cells treated or not treated with TNF-α (Figure S3). These results suggest that SCFA uptake by MCT-1 is essential for SCFA-mediated suppression of inflammatory response in Caco-2 cells. DSS Administration Induced Colonic Inflammation in Mice. As shown in Figure 5A, body weight gain in DSS group was lower than that in control group at 3 days after the start of DSS administration and thereafter (P < 0.05). At the same

Reduction of TNF-α-Induced Activation of NF-κB, ERK, p38 MAPK, JNK, and Syk in Caco-2 Cells by SCFAs. The levels of phosphorylated (i.e., activated) NF-κB p65, Syk, ERK1/2, JNK1/2, and p38 MAPK in Caco-2 monolayer cells incubated with TNF-α for 30 min were higher than in control Caco-2 cells (Figure 3, P < 0.01). Exposure to acetate, propionate, or butyrate decreased enhanced phosphorylation of Syk, ERK1/2, JNK1/2, and p38 MAPK induced by TNF-α. The level of NF-κB p65 phosphorylation in the cells incubated with propionate was lower than that in cells exposed to TNF-α alone. The nominal reductions of NF-κB p65 phosphorylation levels by acetate and butyrate did not achieve the level of statistical significance. Reduction of SCFA-Mediated Suppression of IL-8 Expression in Caco-2 Cells by an MCT-1 Inhibitor. 112

DOI: 10.1021/acs.jafc.7b04233 J. Agric. Food Chem. 2018, 66, 108−117

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Figure 5. Induction of acute colitis in mice by the administration of dextran sodium sulfate. Body weight gain (A), clinical score (B), and hematoxylin and eosin staining of colons (C) in control mice and mice administered with dextran sodium sulfate (DSS) are shown. (A, B) Data are represented as the mean ± SEM of six animals. *P < 0.05 vs control mice. (C) Representative images of colons from a control, healthy mouse (left panel) and a colitic mouse (right panel).

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DISCUSSION A growing body of evidence suggests that intestinal homeostasis is critically important for human health. IBDs are a group of gastrointestinal disorders, such as CD and UC, characterized by chronic intestinal inflammation.41,42 Patients with IBDs suffer from diarrhea, rectal bleeding, abdominal pain, and cramps for long periods.43 Chronic inflammation develops with excessive and uncontrolled production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-8. The present study provides evidence of anti-inflammatory activity of SCFAs, which are major metabolites of intestinal fermentation by microorganisms. Acetate, propionate, and butyrate reduced TNF-α-induced inflammatory signaling and expression levels of IL-8 and IL-6 in intestinal Caco-2 and T84 cells. Similar results were observed in cultures of inflamed colonic segments prepared from colitic mice and exposed to SCFA mixtures. As these effects were sensitive to pharmacological inhibition of MCT-1, anti-inflammatory activity of SCFAs is apparently dependent upon their transport by MCT-1 localized on the apical membrane of intestinal epithelial cells. Although IBD pathogenesis still remains unclear, basic experimental and clinical studies have demonstrated that the inflammatory signaling elicited by TNF-α has an essential role in the development of inflammation. TNF receptor 2 (TNFR2) deficient mice showed resistance to DSS-induced colitis indicated by decreased clinical score and epithelial proliferation.44 In clinical studies, different types of anti-TNF-α agents have shown efficacy in treatments of both UC and CD.45 This evidence suggests that SCFA-mediated suppression of inflammatory signaling and cytokine expression induced by TNF-α could contribute to the prevention of intestinal

period, the clinical score determined from weight loss, diarrhea, and bloody stool in DSS group was higher than that in control group (Figure 5B, P < 0.01). DSS administration induced colonic structure damages characterized by severe lesions in the mucosa, disruption of crypt structure, enhanced infiltration of immune cells into mucosal and submucosal layers, and colonic edema (Figure 5C). MPO was abundantly expressed in neutrophils infiltrated into the inflamed tissues, and this infiltration was potently induced by IL-8. DSS administration increased MPO activity in colonic tissues, indicating neutrophil infiltration (Figure S4). These results confirmed that DSS administration successfully induced colonic inflammation as described previously. Suppression by SCFAs of Expression Levels of ProInflammatory Cytokines in the Colons of Mice with Experimentally Induced Colitis. Expression levels of the pro-inflammatory cytokines CXCL-2 and IL-6 in the colon were drastically increased by DSS administration (Figure 6A and B, P < 0.01). In mice with experimentally induced colitis, cytokine expressions levels in L-, M-, and H-SCFAs treatment groups were lower than those in the control treatment group. Furthermore, in H-SCFAs treatment group, the values were higher than those in L- and M-SCFAs treatment groups. The expression of the anti-inflammatory cytokine IL-10 in the colons was also increased by DSS administration, but it was not influenced by the treatments with SCFAs (Figure 6C). In control mice, neither treatment altered cytokine expression levels significantly (Figure 6A−C, P > 0.05 for all corresponding comparisons). 113

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discrepancy might be caused by the difference in exposure periods of colonic tissues to butyrate between the studies. The induction of regulatory T cell differentiation may require a longer exposure of cells to butyrate than that used in our study. Alternatively, this result may indicate that SCFA-mediated suppression of IL-6 and IL-8 expression in inflamed mouse colons occurred through the direct stimulation of epithelial cells. SCFAs are known to directly interact with intestinal epithelial cells and influence their cellular functions. Although previous studies indicated at least two types of proteins that could mediate such direct interactions, MCT-1 and GPCRs,17,25 it is plausible that in our study, SCFAs were absorbed into intestinal epithelial cells by apical MCT-1 whereupon they suppressed TNF-α-mediated inflammatory signaling, because SCFA-mediated suppression of IL-8 expression was sensitive to an MCT inhibitor. Furthermore, MCT-1 recruitment to the apical membrane is induced through the stimulation of GPR109a, which is activated by butyrate, but not by acetate or propionate.30 The involvement of GPR109a in the butyrate-mediated IL-8 suppression should be therefore examined in a future study. The SCFAs acetate, propionate, and butyrate apparently suppressed TNF-αinduced activation of several signaling molecules examined in our study: NF-κB, ERK1/2, Syk, JNK1/2, and p38 MAPK, although attenuation of NF-κB phosphorylation in TNF-αtreated Caco-2 cells by acetate and butyrate was not statistically significant. Activation of these signaling pathways by TNF-α reportedly promoted IL-8 expression in intestinal cells.46 Although the inflammatory response to TNF-α is likely mediated by TNFR1 and TNFR2, which are known to be expressed in intestinal epithelial cells, TNFR2, rather than TNFR1, is probably more involved in the inflammatory response in intestinal epithelial cells. For example, TNFR2 has been suggested to mediate inflammation-induced epithelial hyperplasia and TNF-α-induced barrier defect in intestinal cells.44 Upon binding of TNF-α to TNFRs, TNFR-associated factors (TRAFs), multifunctional adaptor proteins, recruit additional proteins to form multiple signaling complexes capable of promoting cellular response. Previous studies showed that TRAFs have crucial roles for NF-κB and MAPKs signaling.47 Future studies should resolve the question of whether SCFAs interfere with the interaction of TRAFs with TNFR2 or other signaling molecules in intestinal cells. Based on the observation that suppressive effects of 5 mM acetate, 2.5 mM propionate, and 0.625 mM butyrate were similar, the following rank order of potency to suppress TNFα-induced IL-8 expression can be derived: acetate < propionate < butyrate. This order apparently correlates with the affinities of SCFAs to MCT-148 and physiological concentrations of SCFAs in colonic lumen.37,38 Interestingly, valerate and lactate did not show any suppressive effects on IL-8 production, although lactate can be a substrate of MCT-1,24,48 whereas succinate, a dicarboxylate, did exhibit a suppressive effect. These findings indicate that there is structural specificity of SCFA effects. The mechanisms underlying succinate-mediated suppression of IL-8 production are unknown. It is possible that succinate influences cellular processes through GPR91, which is expressed in the intestinal epithelial cells and senses luminal succinate.49 Somewhat surprisingly, each SCFA individually, as well as their mixtures, exhibited highest efficacies at the lowest tested concentrations in both Caco-2 cells and mouse colons. A similar result was observed in Caco-2 cells stimulated by

Figure 6. Reduction of CXCL-2 and IL-6 mRNA expression levels in mouse colons induced by the administration of dextran sodium sulfate after exposure to SCFA mixtures. (A−C) Colonic tissue collected from control, healthy animals and mice administered with dextran sodium sulfate (DSS) were incubated for 24 h without or with mixtures of SCFAs (acetate-propionate-butyrate 20:10:2.5, H-SCFAs; 10:5:1.25, M-SCFAs; and 5:2.5:0.625, L-SCFAs, where numbers indicate concentration in mM). Expression levels of CXCL-2 (A), IL6 (B), and IL-10 (C) mRNAs were determined by qRT-PCR. Data are represented as the mean ± SEM of six animals. Means without a common letter differ at the following level of statistical significance: P < 0.05.

inflammation. We found that SCFAs did not influence the expression of IL-10, an anti-inflammatory cytokine mainly produced by regulatory T cells, in cultures of both normal and inflamed colonic segments. However, a recent study showed ̈ T cells with 0.1 mM butyrate for 3 that the stimulation of naive days induced their differentiation into regulatory T cells and ameliorated experimental colitis in mice.9 This apparent 114

DOI: 10.1021/acs.jafc.7b04233 J. Agric. Food Chem. 2018, 66, 108−117

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Journal of Agricultural and Food Chemistry Salmonella in a previous study.10 Although the mechanisms underlying these observations are still unclear, basolateral accumulation of SCFAs transported by epithelial cells may have interfered with the suppressive effects of SCFAs at higher concentrations in our experimental conditions. Inagaki et al. demonstrated that mucosal application of 1 mM butyrate enhanced the proliferation of colonic epithelial cells in the absence of butyrate in the basolateral media, but decreased it if basolateral media contained 1 mM butyrate.50 Owing to the lack of constant blood flow, transported SCFAs could accumulate on the basolateral side of cells, thereby reducing effects of higher applied concentrations of SCFAs in our in vitro studies. We also showed that SCFAs suppressed IL-6 expression in intestinal epithelial cells. In agreement with our observations, Iraporda et al. found that propionate and butyrate suppressed flagellin-induced IL-6 expression in Caco-2 cells.51 SCFAs possibly affect intestinal inflammation through multiple mechanisms, including regulation of T cell differentiation in the intestines. Different cell types, such as epithelial cells, macrophages, T cells, and dendritic cells, exist in intestinal mucosa, and they cooperatively regulate inflammatory status. Acetate, propionate, and butyrate have been reported to suppress the inflammatory response in bone marrow-derived macrophages and dendritic cells. Future studies should further elucidate the precise roles of SCFAs in the regulation of inflammatory processes in the colon. In conclusion, the SCFAs acetate, propionate, and butyrate suppressed up-regulation of cellular signaling and expression of IL-8 and IL-6 in TNF-α-stimulated Caco-2 cells and in colons of colitic mice. Activity of MCT-1, located on the apical membranes, was essential for SCFA effects. Our findings, therefore, shed new light on anti-inflammatory actions of SCFAs in the intestines.

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Funding

This research was partially supported by a Grant-in-Aid for Young Scientists (B) (Kakenhi 16K07737). Tran Van Hung is a recipient of a PhD grant from the Ministry of Education and Training, Vietnam. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED CXCL, chemokine CXC motif ligand; CXCR, CXC chemokine receptor; CD, Crohn’s disease; α-CHA, α-cyano-4hydroxycinnamic acid; DSS, dextran sodium sulfate; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde3-phosphate dehydrogenase; GPCR, G protein coupled receptor; HPRT, hypoxanthine-guanine phosphoribosyltransferase; IBD, inflammatory bowel disease; JNK, c-Jun Nterminal kinase; NF-κB, nuclear factor kappa B; MAPK, mitogen-activated protein kinase; MCT-1, monocarboxylate transporter-1; Syk, spleen tyrosine kinase; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TRAF, TNF receptor associated factor; UC, ulcerative colitis; SCFA, short chain fatty acid

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04233.

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REFERENCES

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Clinical scoring system and primer sequences for qRTPCR (PDF) Sensitivity of basal TNF-α-induced increase in IL-8 expression level in Caco-2 and T84 cells to apical shortchain fatty acids, insensitivity of TNF-α-induced increase in IL-8 expression level in Caco-2 cells to MCT-1 inhibitor, and induction of acute colitis in mice by the administration of dextran sodium sulfate (PDF)

AUTHOR INFORMATION

Corresponding Author

*Telephone: 81-82-424-7984; Fax: 81-82-424-7916; E-mail: [email protected]. ORCID

Takuya Suzuki: 0000-0003-3709-543X Author Contributions

T.V.H. and T.S. designed the research; T.V.H. conducted the study and performed statistical analysis; T.V.H. and T.S. analyzed the data and wrote the paper; T.S. had primary responsibility for the final content. Both authors have read and approved the final manuscript. 115

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