Advertisement

Short-Chain Fatty Acids Activate GPR41 and GPR43 on Intestinal Epithelial Cells to Promote Inflammatory Responses in Mice

  • Myung H. Kim
    Affiliations
    Laboratory of Immunology and Hematopoiesis, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana
    Search for articles by this author
  • Seung G. Kang
    Affiliations
    Laboratory of Immunology and Hematopoiesis, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana
    Search for articles by this author
  • Jeong H. Park
    Affiliations
    Laboratory of Immunology and Hematopoiesis, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana
    Search for articles by this author
  • Masashi Yanagisawa
    Affiliations
    Department of Molecular Genetics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas
    Search for articles by this author
  • Chang H. Kim
    Correspondence
    Reprint requests Address requests for reprints to: Chang Kim, PhD, Department of Comparative Pathobiology, 725 Harrison Street, Purdue University, West Lafayette, Indiana 47907 fax: 1-765-494-9830.
    Affiliations
    Laboratory of Immunology and Hematopoiesis, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

    Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana

    Center for Cancer Research, Purdue University, West Lafayette, Indiana
    Search for articles by this author

      Background & Aims

      Short-chain fatty acids (SCFAs), the most abundant microbial metabolites in the intestine, activate cells via G-protein−coupled receptors (GPRs), such as GPR41 and GPR43. We studied regulation of the immune response by SCFAs and their receptors in the intestines of mice.

      Methods

      Inflammatory responses were induced in GPR41−/−, GPR43−/−, and C57BL6 (control) mice by administration of ethanol; 2, 4, 6-trinitrobenzene sulfonic-acid (TNBS); or infection with Citrobacter rodentium. We examined the effects of C rodentium infection on control mice fed SCFAs and/or given injections of antibodies that delay the immune response. We also studied the kinetics of cytokine and chemokine production, leukocyte recruitment, intestinal permeability, and T-cell responses. Primary colon epithelial cells were isolated from GPR41−/−, GPR43−/−, and control mice; signaling pathways regulated by SCFAs were identified using immunohistochemical, enzyme-linked immunosorbent assay, and flow cytometry analyses.

      Results

      GPR41−/− and GPR43−/− mice had reduced inflammatory responses after administration of ethanol or TNBS compared with control mice, and had a slower immune response against C rodentium infection, clearing the bacteria more slowly. SCFAs activated intestinal epithelial cells to produce chemokines and cytokines in culture and mice after administration of ethanol, TNBS, or C rodentium. These processes required GPR41 and GPR43 and were required to recruit leukocytes and activate effector T cells in the intestine. GPR41 and GPR43 activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling pathways in epithelial cells to induce production of chemokines and cytokines during immune responses.

      Conclusions

      SCFAs activate GPR41 and GPR43 on intestinal epithelial cells, leading to mitogen-activated protein kinase signaling and rapid production of chemokines and cytokines. These pathways mediate protective immunity and tissue inflammation in mice.

      Keywords

      Abbreviations used in this paper:

      ATF2 (activating transcription factor 2), C2 (acetate), C3 (propionate), C4 (butyrate), CFU (colony-forming unit), ECs (epithelial cells), ERK (extracellular signal-regulated kinase), FITC (fluorescein isothiocyanate), GPR (G-protein−coupled receptor), IL (interleukin), KO (knock-out), MAPK (mitogen-activated protein kinase), SCFAs (short-chain fatty acids), Th (T-helper cell), TNBS (2, 4, 6-trinitrobenzene sulfonic-acid), WT (wild type)
      The gut immune system normally maintains immune tolerance to harmless antigens but mounts efficient immune responses to infection by pathogens. The factors and mechanisms that regulate immune responses in the intestine remain incompletely understood. The intestine is densely populated with commensal bacteria, which metabolize dietary fibers and other materials in the colon.
      • Ventura M.
      • Turroni F.
      • Canchaya C.
      • et al.
      Microbial diversity in the human intestine and novel insights from metagenomics.
      • Hamer H.M.
      • De Preter V.
      • Windey K.
      • et al.
      Functional analysis of colonic bacterial metabolism: relevant to health?.
      The gut microbiota actively produces a number of immune regulatory metabolites.
      • Jarchum I.
      • Pamer E.G.
      Regulation of innate and adaptive immunity by the commensal microbiota.
      • Sartor R.B.
      Key questions to guide a better understanding of host-commensal microbiota interactions in intestinal inflammation.
      Short-chain fatty acids (SCFAs), such as acetate (C2), propionate (C3), and butyrate (C4) are major end products of gut microbial fermentation and are implicated in the regulation of the gut immune system.
      • Macia L.
      • Thorburn A.N.
      • Binge L.C.
      • et al.
      Microbial influences on epithelial integrity and immune function as a basis for inflammatory diseases.
      SCFAs either positively or negatively affect oxidative burst, degranulation, and phagocytic functions.
      • Le Poul E.
      • Loison C.
      • Struyf S.
      • et al.
      Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation.
      • Brunkhorst B.A.
      • Kraus E.
      • Coppi M.
      • et al.
      Propionate induces polymorphonuclear leukocyte activation and inhibits formylmethionyl-leucyl-phenylalanine-stimulated activation.
      • Menzel T.
      • Luhrs H.
      • Zirlik S.
      • et al.
      Butyrate inhibits leukocyte adhesion to endothelial cells via modulation of VCAM-1.
      SCFAs are used by epithelial cells (ECs) as an energy source
      • Ventura M.
      • Turroni F.
      • Canchaya C.
      • et al.
      Microbial diversity in the human intestine and novel insights from metagenomics.
      • Hamer H.M.
      • De Preter V.
      • Windey K.
      • et al.
      Functional analysis of colonic bacterial metabolism: relevant to health?.
      and regulate their proliferation, differentiation, and apoptosis.
      • Hague A.
      • Elder D.J.
      • Hicks D.J.
      • et al.
      Apoptosis in colorectal tumour cells: induction by the short chain fatty acids butyrate, propionate and acetate and by the bile salt deoxycholate.
      In addition, SCFAs promote mineral absorption, lipid metabolism, mucin production, and expression of antimicrobial peptides.
      • Kles K.A.
      • Chang E.B.
      Short-chain fatty acids impact on intestinal adaptation, inflammation, carcinoma, and failure.
      SCFAs enter cells through diffusion or monocarboxylate transporters and solute transporters.
      • Thwaites D.T.
      • Anderson C.M.
      H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine.
      SCFAs also activate cells through cell-surface G-protein−coupled (GPR) receptors, such as GPR41 and GPR43.
      • Brown A.J.
      • Goldsworthy S.M.
      • Barnes A.A.
      • et al.
      The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids.
      These 2 receptors are differentially expressed by ECs, adipocytes, and phagocytes, and regulate diverse cellular functions.
      • Tazoe H.
      • Otomo Y.
      • Kaji I.
      • et al.
      Roles of short-chain fatty acids receptors, GPR41 and GPR43 on colonic functions.
      • Xiong Y.
      • Miyamoto N.
      • Shibata K.
      • et al.
      Short-chain fatty acids stimulate leptin production in adipocytes through the G protein-coupled receptor GPR41.
      • Samuel B.S.
      • Shaito A.
      • Motoike T.
      • et al.
      Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41.
      • Maslowski K.M.
      • Vieira A.T.
      • Ng A.
      • et al.
      Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43.
      • Sina C.
      • Gavrilova O.
      • Forster M.
      • et al.
      G protein-coupled receptor 43 is essential for neutrophil recruitment during intestinal inflammation.
      The functions of SCFAs and their receptors in regulation of immune responses are poorly understood. We investigated the functions of SCFAs and their receptors in the regulation of immune responses to immunological challenges including breach of the gut barrier function; 2, 4, 6-trinitrobenzene sulfonic-acid (TNBS) treatment; and infection with Citrobacter rodentium. We provide evidence that SCFAs condition gut ECs to mount prompt immune responses to all immunological challenges in a GPR41 and GPR43-dependent manner. SCFAs and their receptors promote acute inflammatory responses in the intestine for protective immunity and tissue inflammation.

      Materials and Methods

       Animals

      All experiments with animals in this study were approved by the Purdue Animal Care and Use Committee. C57BL/6 mice were from Harlan Laboratories (Indianapolis, IN). CD45.1 C57BL/6 mice were from The Jackson Laboratory (Bar Harbor, ME). GPR43−/− C57BL/6 mice were from Deltagen (San Mateo, CA), and GPR41−/− C57BL/6 mice were described previously.
      • Xiong Y.
      • Miyamoto N.
      • Shibata K.
      • et al.
      Short-chain fatty acids stimulate leptin production in adipocytes through the G protein-coupled receptor GPR41.

       Induction of Intestinal Inflammation with Ethanol and TNBS

      Mice were starved overnight and received 100 μL 50% ethanol in the rectum using a round-tip needle. Intestinal inflammation with TNBS was induced as described previously.
      • Wirtz S.
      • Neufert C.
      • Weigmann B.
      • et al.
      Chemically induced mouse models of intestinal inflammation.
      Mice were monitored daily for changes in body weight, signs of illness, and stool scores. Stool consistency scores were measured as follows: 0 = normal stool; 1 = soft stool; 2 = diarrhea; 3 = diarrhea and anal bleeding. Mice were sacrificed 1 day after ethanol treatment or 3 days after TNBS rectal administration. The detailed method to score tissue inflammation is described in the Supplementary Material.

       Infection with C rodentium

      Wild-type (WT), GPR41−/−, and GPR43−/− mice were infected with C rodentium (DBS100, 109 colony-forming unit [CFU]/mouse) via oral gavage as described previously.
      • Simmons C.P.
      • Clare S.
      • Ghaem-Maghami M.
      • et al.
      Central role for B lymphocytes and CD4+ T cells in immunity to infection by the attaching and effacing pathogen Citrobacter rodentium.
      For infection of mice after C2 administration, mice were fed with C2 (200 mM, pH 7.5) in drinking water for 4 weeks and infected with a higher dose (1010 CFU/mouse) of C rodentium. When indicated, mice were injected intraperitoneally with antibodies (50 μg/injection for each antibody) to neutralize interleukin (IL)-6 (MP5-20F3 on day 0 and 2) and deplete neutrophils (RB6-8C5 on day −2 and 0). Mice were monitored for weight change and stool consistency. Mice were sacrificed at indicated time points, and the C rodentium load in feces and tissues was determined as described in the Supplementary Material. Indicated tissues were examined for frequencies of neutrophils and T-cell subsets by flow cytometry.

       Bone Marrow Reconstitution

      Recipient mice were exposed to 960 cGy ionizing radiation and were reconstituted with donor marrow cells (1 × 107/mouse) injected into a tail vein. The mice were kept for 8 weeks for hematopoietic reconstitution and were sensitized and administered with TNBS or orally infected with C rodentium (109 CFU/mouse). CD45.1 mice were used as recipients when GPR41−/− (CD45.2) and GPR43−/− (CD45.2) mice were used as marrow donors. When GPR41−/− and GPR43−/− mice were used as the recipients, CD45.1 mice were used as the marrow donors. The marrow reconstitution efficiency in these animals was >95%.

       Immunohistochemistry

      For immunofluorescence staining, 6-μm–thick frozen and paraffin colon sections were stained with rabbit polyclonal antibodies to ZO-1 (Invitrogen, Carlsbad, CA) and phospho-p44/42 MAPK (Erk1/2) (Cell Signaling Technology, Danvers, MA), respectively. Slides were further stained with fluorescently labeled polyclonal anti-rabbit IgG antibodies (Invitrogen). Tissue sections were stained with anti–Gr-1 (RB6-8C5) and Hoechst 33342 (Invitrogen) when indicated, and the images were acquired with a Leica SP5 II confocal microscope.

       Microarray and Real-Time Polymerase Chain Reaction

      Colon tissues were frozen in liquid nitrogen and ground using mortar and pestle. The array data were analyzed as described
      • Kang S.G.
      • Park J.
      • Cho J.Y.
      • et al.
      Complementary roles of retinoic acid and TGF-beta1 in coordinated expression of mucosal integrins by T  cells.
      and deposited at: http://www.ncbi.nlm.nih.gov/geo (GSE36569). Quantitative real-time polymerase chain reaction was performed as described previously.
      • Kang S.G.
      • Park J.
      • Cho J.Y.
      • et al.
      Complementary roles of retinoic acid and TGF-beta1 in coordinated expression of mucosal integrins by T  cells.

       Gut Permeability

      Mice fasted overnight, received intragastrically with fluorescein isothiocyanate (FITC)–conjugated dextran (0.2 mg/g body weight (g) mean molecular weight 3000–5000; Sigma-Aldrich, St Louis, MO) using a round-tip feeding needle. Mice were sacrificed 3 h later, and the FITC-dextran concentration in the plasma was determined with a fluorescent microplate reader (excitation at 485 nm and emission at 535 nm; BioTek Synergy HT; BioTek, Winooski, VT).

       In vitro Culture of Colonic ECs

      Lamina propria cells and colonic ECs (viability and purity >95% based on CD45 negativity) were isolated as described previously.
      • Kang S.G.
      • Park J.
      • Cho J.Y.
      • et al.
      Complementary roles of retinoic acid and TGF-beta1 in coordinated expression of mucosal integrins by T  cells.
      • Booth C.
      • O'Shea J.A.
      Isolation and culture of intestinal epithelial cells.
      For quantitative real-time polymerase chain reaction analysis of GPR41 and GPR43, CD326+ cells were selected with Miltenyi magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Colonic ECs (2 × 105 cells/well) were cultured for 6 days, and C2 (10 mM) or C3 (1 mM) was added during this culture period. These cells were further activated with lipopolysaccharide (Sigma-Aldrich; 1 μg/mL) or a commensal bacterial extract (20 μg/mL) for 24 h. Alternatively, colonic ECs, established for 3 days, were treated with kinase inhibitors and SCFAs for 3 days. Please see the Supplementary Material for the experiment with the inhibitors.

       Flow Cytometry

      Flow cytometry was performed as described previously.
      • Kang S.G.
      • Park J.
      • Cho J.Y.
      • et al.
      Complementary roles of retinoic acid and TGF-beta1 in coordinated expression of mucosal integrins by T  cells.
      For neutrophils, tissue cells were stained with antibodies to Gr-1 (RB6-8C5) and 7/4 (Ly-6B.2). For phosphorylation of extracellular signal-regulated kinase (ERK) and ATF2 in ECs, cells were fixed and permeabilized with perm III buffer (BD Bioscience, San Diego, CA) and stained with antibodies to phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) or phospho-ATF-2 (Thr71) (Cell Signaling Technology). Donkey anti-rabbit IgG-FITC (BioLegend, San Diego, CA) was used as the secondary antibody.

       Statistical Analysis

      Student's t test, Mann-Whitney test, and one-way analysis of variance were used to determine significance of the differences between groups. P values ≤.05 were considered significant.

      Results

       Mice Deficient With GPR41 or GPR43 Are Defective in Mounting the Normal Inflammatory Response After an Ethanol-Induced Breach of the Gut Barrier Function

      To determine the functions of SCFAs and their receptors in regulation of acute antibacterial responses, we temporarily breached the gut barrier function through rectal administration of ethanol into WT, GPR41−/−, and GPR43−/− mice. As expected, the ethanol treatment induced a transient decrease in the weight of WT mice at 24 h after the treatment (Figure 1A). However, the 2 knockout (KO) mouse strains were significantly less affected by the treatment. Gross examination revealed that only WT mice, but not the 2 KO strains, had apparent shortening and thickening of the colon (Figure 1B). The ethanol treatment induced infiltration of neutrophils within the colon tissue (Figure 1C). In this regard, the neutrophil frequency in the colonic lamina propria was abnormally low in the SCFA-receptor KO mice compared with WT mice. In line with this, GPR41−/− and GPR43−/− mice, unlike WT mice, did not display severe histological changes and leukocyte infiltration in the mucosa and submucosa (Supplementary Figure 1).
      Figure thumbnail gr1
      Figure 1Hypo-inflammatory responses after ethanol-induced breach of intestinal barrier function in GPR41−/− and GPR43−/− mice. (A) Weight change after treatment with 50% ethanol in the rectum (n = 10−15/group). (B) Gross appearance and length of the colon after ethanol treatment (n = 14−15/group). (C) Neutrophil infiltration in the colon after ethanol treatment. Percent neutrophil frequency among lamina propria cells is shown. (D) Expression of ZO-1 and GR-1 in the colon and changes in the gut barrier function during the inflammation. The gut barrier function was determined based on FITC-dextran leakage into the blood after the ethanol treatment (n = 10−12/group). (E) Levels of colon tissue-associated commensal bacteria after ethanol treatment (n = 10−13/group). Bacterial 16S ribosomal RNA gene levels in the colon tissue were examined by quantitative polymerase chain reaction. All data were obtained at 24 h after ethanol treatment. Pooled data from 3 independent experiments are shown. Significant differences (P < .05) from untreated WT mice or ethanol-treated WT mice.∗∗
      Gut permeability is increased during immune responses.
      • Boirivant M.
      • Amendola A.
      • Butera A.
      • et al.
      A transient breach in the epithelial barrier leads to regulatory T-cell generation and resistance to experimental colitis.
      Unlike WT mice, GPR41−/− and GPR43−/− mice did not lose ZO-1 expression and had relatively mild tissue infiltration with Gr-1+ neutrophils after the ethanol treatment (Figure 1D). Gut barrier function was determined based on FITC-dextran leakage from the colonic lumen into blood circulation. Compared with WT mice, the 2 KO strains had smaller gut permeability changes. As the gut permeability increased, so did the bacterial cells within the tissue. Again, GPR41−/− and GPR43−/− mice had smaller increases in tissue bacteria compared with WT mice (Figure 1E).
      To gain more insight into the inflammatory response regulated by the SCFA-receptor signals, we performed an Affymetrix microarray gene expression study. Many genes induced in WT mice were significantly less induced in the 2 SCFA-receptor KO strains (Figure 2). Interestingly, there is a striking similarity in overall gene expression between GPR41−/− and GPR43−/− mice (Figure 2A). GPR41−/− and GPR43−/− mice were defective in up-regulation of certain CCL and CXCL chemokines and inflammatory cytokines (ie, IL-1β, IL-6, and IL-11) (Figure 2B). Also defective was expression of metalloproteinases, including MMP7, MMP9, and MMP10. The defective expression of chemokines, cytokines, and metalloproteinases in SCFA-receptor KO mice was confirmed with quantitative real-time polymerase chain reaction (Figure 2C). These results indicate that SCFA-receptor KO mice are defective in up-regulating key inflammatory mediators in the intestine.
      Figure thumbnail gr2
      Figure 2Defective induction of inflammation-associated genes in GPR41−/− and GPR43−/− mice after ethanol-induced breach of intestinal barrier function. (A) Genes differentially expressed in the colon tissues of WT vs SCFA-receptor KO mice after the ethanol treatment. Microarray data are shown in a multi-plot format showing gene expression differences between WT and KO mice deficient with GPR41 or GPR43. Mean values obtained from 2 independent experiments are shown. (B) Treeviews of selected chemokine and cytokine genes up- or down-regulated after the ethanol treatment. (C) Quantitative real-time polymerase chain reaction analysis of selected genes. All of the data were obtained at 24 h after ethanol treatment. Pooled data from 3 independent experiments (total n = 3) are shown. Significant differences (P < .05) from untreated WT mice or ethanol-treated WT mice.∗∗

       Mice Deficient With GPR41 or GPR43 Are Inefficient in Mounting the Inflammatory Response to TNBS

      We further examined the response to TNBS. The TNBS challenge decreased body weight in WT mice by approximately 20% 3 days after the rectal challenge (Figure 3A). The same treatment decreased body weight by only about 10% in SCFA-receptor KO mice. Only WT, but not the KO, mice had soft stool on day 2 (Figure 3B). WT mice had considerable shortening of the colon (∼20% decrease) as expected, but the colon of GPR41−/− and GPR43−/− mice was significantly less affected (∼10% decrease) (Figure 3C). Induction of IL-6, IL-12, and T-helper cell (Th) 1−associated genes (interferon gamma and T-bet) was defective in both GPR41−/− and GPR43−/− mice (Figure 3D and Supplementary Figure 2A). In line with this, the frequency of Th1 cells was decreased in the small intestine, colon, and mesenteric lymph node of the KO mice (Supplementary Figure 2B). In addition, induction of neutrophil infiltration and expression of CXCL1, CXCL2, and CCL2 in the colon were abnormally low in GPR41−/− and GPR43−/− mice (Figure 3E and F). Histologic examination of the distal colon revealed more severe leukocyte infiltration, mucosa hyperplasia, and tissue damage in WT mice than the KO mice (Supplementary Figure 3). These results indicate that the 2 SCFA receptors play positive roles in mounting the acute inflammatory response to TNBS.
      Figure thumbnail gr3
      Figure 3Abnormally low immune responses to TNBS in GPR41−/− and GPR43−/− mice. (A) Weight change after the TNBS treatment. (B) Stool consistency scores. (C) Colon length. (D) Levels of IL-6 in the blood or conditioned media of colonic ECs prepared from TNBS-treated mice. (E) Infiltration of phagocytes in the colon. (F) Expression of chemokines in the distal colon. Quantitative real-time polymerase chain reaction was performed. All data were obtained on day 3 after TNBS treatment. Combined (n = 12−30/group) or representative data from 4 independent experiments are shown. Significant differences (P < .05) from untreated WT mice or TNBS-treated WT mice.∗∗

       GPR41 and GPR43 Signals Are Required for Undelayed Induction of the Immune Response to C rodentium

      We used a C rodentium infection model to determine the role of SCFA receptors in mounting immune responses to bacterial pathogens. GPR41−/− and GPR43−/− mice were more susceptible than WT mice based on weight change and stool consistency scores (Figure 4A and B). Clearance of C rodentium from the intestine was delayed in the KO mice (Figure 4C). There was increased translocation of the pathogen into the liver and spleen of the KO mice. The KO mice had low responses in blood IL-6 (Supplementary Figure 4), neutrophil recruitment (Figure 4D), and frequencies of Th1 and Th17 cells in the colon (Figure 4E). We examined the expression kinetics of inflammatory cytokines (ie, IL-6, IL-17A, and IL-12), and interferon gamma and chemokines (CXCL1 and CXCL2) and found that induction of these cytokines and chemokines was delayed by 1−2 weeks (Supplementary Figure 5A and B). We next examined the gut permeability change regulated by SCFA receptors. There was no difference in gut permeability or expression of tight junction protein genes (ZO-1 and Occludin) between WT and any of the 2 SCFA receptor KO mice in the absence of any immunological challenges (Supplementary Figure 6). However, on infection with C rodentium, GPR41, or GPR43 KO mice had a delayed kinetics in gut permeability change (Figure 4F). Although the tissue bacteria level was lower at early time points (before 14 days) in the KO mice compared with WT mice, it was considerably higher at later time points, reflecting increased pathogen invasion and/or bacterial translocation into the tissue.
      Figure thumbnail gr4
      Figure 4Delayed immune responses and increased susceptibility of GPR41−/− and GPR43−/− mice to C rodentium infection. (A) Weight change after C rodentium infection. (B) Stool consistency scores. (C) C rodentium loads (CFU/g feces or tissue) in feces and indicated organs. (D) Neutrophil infiltration in the colon during infection. (E) Th1 and Th17 cell frequencies in the colon at 2 weeks post infection. (F) Gut permeability change and tissue-associated eubacteria after infection. Pooled data obtained from 3 independent experiments are shown (10−15/group for AE; 4−5/group for F). Significant differences (P < .05) between WT and GPR41 KO mice or WT and GPR43 KO mice.∗∗

       Immune Response Promoted by SCFAs Is Required for Timely Clearance of C Rodentium

      We next examined the effect of an increased C2 concentration on the antibacterial immune response. For this, we fed mice with C2 and examined the kinetics of the antibacterial response to C rodentium. C2-fed mice suffered less than WT mice from the infection based on weight change and stool consistency (Figure 5A) and the intestinal C rodentium load (Figure 5B). C2 administration accelerated the expression of IL-6, CXCL1, and CXCL2 (Figure 5C). Also increased upon the C2 administration was recruitment of neutrophils (Figure 5D) and Th17 cells (Figure 5E) in the large bowel (cecum). C2 administration somewhat accelerated the gut permeability change (Figure 5F). C2 administration accelerated the overall immune response during the infection with C rodentium.
      Figure thumbnail gr5
      Figure 5C2 administration accelerates the immune response to C rodentium infection. (A) Weight change and stool consistency scores of WT and C2-fed mice after C rodentium infection. (B) Fecal C rodentium load. (C) Expression of IL-6, CXCL1, and CXCL2 in the cecum. (D) Neutrophils in colon. (E) Th17 cells in the colon. (F) Gut permeability change. Some mice were injected twice with antibodies to neutralize IL-6 (day 0 and 2) and deplete neutrophils (day −2 and 0). Pooled data obtained from 3 independent experiments are shown (n = 10−15/group). Significant differences (P < .05) between WT and WT-C2 () or WT and WT-Ab-Ly6G/IL6 (∗∗).
      To assess the importance of the immune response promoted by SCFAs, we included animal groups where key immune components (IL-6 and neutrophils) were neutralized or depleted just before or after the infection with C rodentium. As expected, the antibody-injected mice were more susceptible than control mice to C rodentium (Figure 5A and B), demonstrating the important roles of IL-6 and neutrophils in induction of antibacterial immunity. The antibody treatment delayed the induction of IL-6 and neutrophil-attracting chemokines (Figure 5C). In line with this, the increase of neutrophils (Figure 5D) and Th17 cells (Figure 5E) in the large bowel was significantly delayed with the antibody treatment. The boosting effect of C2 administration was mostly abolished by the antibody treatment. Overall, the immune responses promoted by SCFAs are critical for timely clearance of pathogens.
      We also examined the impact of the antibody-induced delay in immune responses on gut permeability change. This early injection of antibodies somewhat suppressed the increase in gut permeability at an early time point (day 1−12) (Figure 5F). At later time points (day 15 and later), however, a greater change in gut permeability occurred in the antibody-injected animals (Figure 5F). The hyper-gut permeability at later time points is in line with the higher C rodentium load and the subsequently increased cytokine/chemokine response to the bacteria (Figure 5B and C). Overall, these results suggest that the gut permeability change is induced by the immune response promoted by the SCFA signal.
      SCFA receptor deficiency can affect the commensal bacteria profile in the gut to indirectly affect the immune response. To determine the possibility, we examined the levels of 7 bacterial groups in the gut of GPR41−/− and GPR43−/− mice. We found no significant change in the gut commensal bacteria profile (Supplementary Figure 7).

       Roles of SCFA Receptors Expressed by Bone Marrow vs Non−Marrow-Derived Cells in Promoting the Antibacterial Immune Response

      To determine the roles of SCFA receptors expressed by marrow vs non−marrow-derived cells, we prepared bone marrow chimera mice with WT recipients and GPR41−/− or GPR43−/− marrow donors or vice versa. At 8 weeks post-transplantation, these mice were infected with C rodentium, and clinical scores and immune responses after the infection were examined (Figure 6). WT mice reconstituted with WT marrow (WT→WT) or KO marrow (41KO→WT or 43KO→WT) did not lose weight significantly after infection. In contrast, the KO recipient mice, reconstituted with WT marrow (WT→41KO or WT→43KO), lost weight significantly (Figure 6A). Stool consistency score and C rodentium load were significantly higher in WT→41KO mice and WT→43KO mice compared with WT→WT mice (Figure 6B and C). In line with the defective clearance of C rodentium, frequencies of Th17 cells and neutrophils were lower in the colon of WT→41KO mice and WT→43KO mice compared with other mice (Figure 6D and E). These data indicate that the SCFA receptors expressed by non−marrow-derived cells play bigger roles than those of marrow-derived cells in promoting the antibacterial responses.
      Figure thumbnail gr6
      Figure 6Differential roles of the SCFA receptors expressed by non-bone marrow vs marrow-derived cells in promoting the immune response to C rodentium. (A) Weight change after C rodentium infection in bone marrow chimera mice. (B) Stool consistency scores. (C) Fecal C rodentium load. (D) Th17 cells in the colon. (E) Infiltration of the colon by neutrophils. Chimera mice were infected at 8 weeks after the bone marrow transfer (donor→host). (F) Expression of GPR41 and GPR43 messenger RNA by Gr-1+ neutrophils, CD326+ ECs, CD19+ B cells, and CD4+ T cells. Pooled data (8−10 mice/group) obtained from 2−3 independent experiments are shown. Significant differences from WT→WT mice or between indicated groups.∗∗
      We also examined the inflammatory response to TNBS in the bone marrow chimera mice (Supplementary Figure 8). The inflammatory response to TNBS was less severe in WT→41KO and WT→43KO compared to WT→WT, 41KO→WT, and 43KO→WT mice based on the weight change (Supplementary Figure 8A), stool consistency scores (Supplementary Figure 8B), gross appearance and length of the colon (Supplementary Figure 8C), neutrophil frequency (Supplementary Figure 8D), and Th1 cell frequency (Supplementary Figure 8E). These data further support the positive roles of GPR41 and GPR43 expressed by non−marrow-derived cells in promoting the acute inflammatory response.

       SCFAs Condition ECs for Optimal Production of Chemokines and Inflammatory Cytokines in a MEK-ERK−Dependent Manner

      The bone marrow chimera study in Figure 6 reveals that non−marrow-derived cells play important roles in mediating the SCFA-dependent inflammatory response. Enteroendocrine cells and colonic enterocytes express GPR43 and GPR41.
      • Wells J.M.
      • Loonen L.M.
      • Karczewski J.M.
      The role of innate signaling in the homeostasis of tolerance and immunity in the intestine.
      • Wullaert A.
      Role of NF-kappaB activation in intestinal immune homeostasis.
      • Barnes M.J.
      • Powrie F.
      Regulatory T cells reinforce intestinal homeostasis.
      We confirmed that both GPR41 and GPR43 messenger RNAs were highly expressed by CD326+ (epithelia cell adhesion molecule) ECs in the intestine (Figure 6F). We determined the possibility that SCFAs condition ECs for more efficient production of chemokines and inflammatory cytokines in response to bacterial products. We established primary colonic ECs in vitro and activated them for 24 h with lipopolysaccharide or a gut commensal bacterial extract. Interestingly, small but detectable induction of IL-6 expression in response to C2 or C3 alone was observed (Figure 7A and Supplementary Figure 9). In addition, expression of IL-6, CXCL1, and CXCL10 in response to the bacterial products was increased in the presence of C2 or C3. The induction was abolished when colonic ECs were pretreated with pertussis toxin, which is an inhibitor of G0/i-coupled receptors, such as GPR41 and GPR43 (Supplementary Figure 9). The ECs, isolated from GPR41−/− or GPR43−/− mice, were largely unresponsive to C2 or C3 in expression of the cytokines (Figure 7A) and chemokines beyond basal levels (Figure 7B).
      Figure thumbnail gr7
      Figure 7SCFA-dependent expression of inflammatory chemokines and cytokines is dependent on epithelial GPR41 and GPR43 and the MEK-ERK pathway. (A) Production of inflammatory cytokines by WT, GPR41−/−, and GPR43−/− colonic ECs pretreated with SCFAs and lipopolysaccharide (LPS). (B) Expression of chemokines by colonic ECs in response to SCFAs and LPS. (C) SCFAs induced phosphorylation of ERK in ECs. (D) Effects of signaling molecule inhibitors on SCFA-dependent expression of IL-6. Commonly used concentrations of the inhibitors without significant off-target effects were used. (E) Defective SCFA-induced phosphorylation of ERK in the colon of GPR41−/− and GPR43−/− mice 5 days after infection with C rodentium. (F) SCFA-dependent phosphorylation of ATF2 in WT, GPR41−/−, and GPR43−/− ECs. Pooled data obtained from 3 independent experiments (n = 3) are shown. Significant differences (P < 0.05) from C2/C3-untreated (AC, F) or between control and inhibitor-treated groups (D) for C2∗∗ or C3∗∗∗.
      We studied the intracellular pathways important for SCFA-dependent expression of the cytokines. SCFAs activate the MEK-ERK pathway in neutrophils.
      • Vinolo M.A.
      • Ferguson G.J.
      • Kulkarni S.
      • et al.
      SCFAs induce mouse neutrophil chemotaxis through the GPR43 receptor.
      We confirmed that C2 and C3 induced phosphorylation of ERK in primary colonic ECs (Figure 7C). The SCFA-dependent phosphorylation of ERK did not occur in GPR41−/− and GPR43−/− ECs (Figure 7C), suggesting that ERK activation is induced by the SCFA signals through GPR41 and GPR43. This ERK activation was suppressed by the following inhibitors of ERK: PD98059 (a MEK1 inhibitor) and U0126 (a MEK1/2 inhibitor) (Figure 7D). These inhibitors also suppressed the expression of IL-6, CXCL1, and CXCL2 induced in response to C2 or C3 (Supplementary Figure 10). Also, p38 MAPK is implicated in the SCFA receptor signaling in a breast cancer cell line.
      • Yonezawa T.
      • Kobayashi Y.
      • Obara Y.
      Short-chain fatty acids induce acute phosphorylation of the p38 mitogen-activated protein kinase/heat shock protein 27 pathway via GPR43 in the MCF-7 human breast cancer cell line.
      SB203508 (a p38 inhibitor) was suppressive on induction of IL-6 and chemokines (Figure 7D and Supplementary Figure 10). However, a c-Jun N-terminal kinases/c-Jun N-terminal kinase inhibitor, SP600125, was ineffective. We confirmed, using an immunohistochemistry technique, that activation of ERK was greatly enhanced with C2 administration through drinking water in the colon of WT, but not GPR41 or GPR43 KO, mice infected with C rodentium (Figure 7E). Activating transcription factor 2 (ATF2) is activated downstream of ERK and MAPK pathways and involved in expression of inflammatory cytokines and chemokines.
      • Lau E.
      • Ronai Z.A.
      ATF2—at the crossroad of nuclear and cytosolic functions.
      We examined if C2 and C3 would activate ATF2 in a GPR41/GPR43-dependent manner in ECs. ATF2 activation was induced by C2 and C3, but this activation did not occur in GPR41 or GPR43 KO cells (Figure 7F).

      Discussion

      The mechanism of action for probiotics, prebiotics, and SCFAs to promote immunity in the gut is incompletely understood.
      • Sanz Y.
      • Nadal I.
      • Sanchez E.
      Probiotics as drugs against human gastrointestinal infections.
      We studied the roles of SCFA receptors in regulating immune responses in the intestine. The results identified positive roles of SCFAs and their receptors in preparing ECs to promptly mount immune responses during immunological challenges.
      Our study demonstrated that SCFA signals are required for mounting acute inflammatory responses in the colon after ethanol treatment. Expression of key mediators of inflammatory responses, including chemokines and cytokines, was defective in the absence of GPR41 and GPR43. There have been mixed reports regarding the roles of SCFAs in regulation of chronic gut inflammation. Some reported that parenteral administration of butyrate increased gut expression of IL-1β and IL-6.
      • Milo L.A.
      • Reardon K.A.
      • Tappenden K.A.
      Effects of short-chain fatty acid-supplemented total parenteral nutrition on intestinal pro-inflammatory cytokine abundance.
      Others reported an anti-inflammatory role of butyrate.
      • Saemann M.D.
      • Bohmig G.A.
      • Osterreicher C.H.
      • et al.
      Anti-inflammatory effects of sodium butyrate on human monocytes: potent inhibition of IL-12 and up-regulation of IL-10 production.
      Contradictory roles of GPR43 were reported. One group reported increased, and the other group reported decreased, dextran sodium sulfate-induced inflammation in the colon of GPR43−/− mice.
      • Maslowski K.M.
      • Vieira A.T.
      • Ng A.
      • et al.
      Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43.
      • Sina C.
      • Gavrilova O.
      • Forster M.
      • et al.
      G protein-coupled receptor 43 is essential for neutrophil recruitment during intestinal inflammation.
      Our results indicate that both delayed and exaggerated immune responses can occur if SCFA signals are deficient. The exaggerated immune responses can occur at later time points and can be caused by delayed immune responses and subsequently increased bacterial load in the intestinal tissue. Different interpretations are likely, depending on the experimental models and time points.
      The TNBS model is a good method to study acute immune responses to a defined antigen (ie, hapten) in the intestine. GPR41−/− and GPR43−/− mice experienced abnormally low inflammatory responses in the colon, as evidenced by low induction of inflammatory chemokines and cytokines and leukocyte infiltration. In addition, mice deficient with GPR41 or GPR43 could not mount a normal Th1 response to TNBS. The TNBS study corroborates the conclusion from the ethanol treatment study that SCFA signals are required for optimal acute inflammatory responses in the gut.
      Utilizing an infection model with C rodentium that examines immune responses occurring during a 4-week period, we found that mice deficient with GPR41 or GPR43 failed to induce the acute inflammatory response to clear the pathogen at early time points. This would lead to delayed clearance and increased invasion of the pathogen into tissues. On the other hand, in vivo C2 administration facilitated clearance of the pathogen. Induction of inflammatory cytokines and chemokines in the GPR41 or GPR43 KO mice was significantly delayed. Consistently, neutrophil infiltration and the Th17 response to bacteria were also delayed. These results suggest that the early inflammatory response promoted by SCFA signals plays a beneficial role in clearance of pathogens.
      SCFA receptors are differentially expressed by ECs, mast cells, phagocytes, and other cell types in the intestine.
      • Wells J.M.
      • Loonen L.M.
      • Karczewski J.M.
      The role of innate signaling in the homeostasis of tolerance and immunity in the intestine.
      • Wullaert A.
      Role of NF-kappaB activation in intestinal immune homeostasis.
      CD326+ ECs express both GPR41 and GPR43 at high levels. Neutrophils express GPR43, but not GPR41, and T and B cells do not express the receptors. Along with the results from the bone marrow reconstitution study, this indicates that ECs are the most likely cells to mediate the observed SCFA effects. This interpretation is further supported by the results that SCFAs activate colonic ECs in a GPR41/43-dependent manner and condition the ECs to effectively respond to bacterial products. The results indicate that timely expression of chemokines and inflammatory cytokines by colonic ECs requires activation of both GPR41 and GPR43 by SCFAs. Although not significantly expressing GPR41, the GPR43-expressing phagocytes can contribute to the overall immune responses regulated by SCFAs. Additional cell types can also mediate the SCFA effects, and this is a subject of importance for future studies. As an intracellular mechanism, we found that the MEK-ERK and p38 MAPK pathways and the ATF2 transcription factor are activated by SCFA signals downstream of GPR41 or GPR43 in gut ECs. Activation of these pathways is required for normal expression of inflammatory cytokines and chemokines by ECs in response to SCFAs.
      The reduced acute immune response in the SCFA-receptor KO mice is mainly due to defective SCFA signaling in ECs. Induction of chemokines and cytokines in ECs by SCFAs in vitro in a GPR41/43-dependent manner supports this interpretation. Our results indicate that the abnormally low cytokine and neutrophil response is the primary reason for the ineffective clearance of pathogens in the GPR41/43 KO mice. Our results ruled out the possibilities that SCFAs increase gut permeability in a steady condition, and that the low immune response would be due simply to reduced gut permeability and bacterial translocation. Gut permeability was even increased in GPR41/43 KO mice at later time points after infection due to the delayed clearance of pathogens and increased antibacterial response. Gut permeability change is an integral part of the overall immune response, which is positively regulated by SCFAs and their receptors through immune responses. In sum, our study revealed novel functions of SCFAs and their receptors in promoting acute inflammatory responses (Supplementary Figure 11), which is beneficial for mounting the immune response to pathogens, but can mediate tissue inflammation.

      Acknowledgments

      The authors thank B. Ulrich and F. Chu (Purdue University) for their excellent assistance and Dr. B. Vallance (University of British Columbia) for providing a C rodentium strain.

      Supplementary Methods

       In vitro Culture of Colonic ECs

      Colonic ECs (2 × 105 cells/well) established in 48-well plates coated with Matrigel basement membrane matrix (BD Bioscience, San Diego, CA). C2 (10 mM) or C3 (1 mM) were activated with lipopolysaccharide (Sigma-Aldrich; 1 μg/mL) or commensal bacterial extract (20 μg/mL) for 24 h. The commensal extract was prepared from the cecal content of 8-week-old C57BL/6 mice with bead beating of the fecal suspension layer. Alternatively, colonic ECs, established for 3 days, were treated with kinase inhibitors (PD98059, U0126, SB203580, and SP600125; Enzo Life Sciences, Inc., Farmingdale, NY) for 1 h before treatment with SCFAs for 3 days. Phosphorylation of ERK in SCFA-treated ECs was examined by flow cytometry as described here. Concentrations of indicated cytokines in serum and conditioned medium were measured in triplicate with a Luminex assay (Luminex Corporation, Austin, TX) or an enzyme-linked immunosorbent assay method. SCFA-treated ECs were harvested and examined by quantitative real-time polymerase chain reaction for expression of chemokines and cytokines.

       Scoring of Intestinal Inflammation

      Histological changes in the distal colon after rectal administration with 50% ethanol with or without TNBS (Sigma-Aldrich). Colon tissues in paraffin were cut into 6-μm−thick sections and stained with H&E. Sections were semi-quantitatively graded on a 0−4 scale based on pathological features, including the extent of leukocyte infiltration, crypt elongation/abscess, bowel wall thickening, mucosa hyperplasia, and ulceration: 0 = no evidence of inflammation; 1 = low level of leukocyte infiltration in <10% high power fields (hpf), no structural changes observed; 2 = moderate leukocyte infiltration in 10%−25% hpf, crypt elongation, and bowel wall thickening; 3 = high level of leukocyte infiltration in 25%−50% hpf, bowel wall thickening and mucosa hyperplasia; and 4 = severe leukocyte infiltration in >50% hpf, crypt elongation, bowel wall thickening, mucosa hyperplasia, and ulceration.

       Bacterial 16S Ribosomal RNA Gene Analysis

      Colon tissues, washed to completely remove the luminal content and feces, were frozen in liquid nitrogen and ground using mortar and pestle. Total DNA was isolated from colon tissues and fecal pellets using QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA). For total eubacteria in tissues, a pair of primers was used to amplify the 16S ribosomal RNA (rRNA) gene sequence conserved in all commensal bacteria. The polymerase chain reaction was performed for 40 cycles (95°C for 30 s and 63°C for 45 s) using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, Logan, UT). Tissue-associated eubacteria levels based on the Ct values of the amplified conserved 16S rRNA gene sequence were normalized with the signal of amplified mouse genomic Actb (β-actin) gene in each sample. The abundance of specific bacterial groups was measured with group-specific 16S rRNA gene primers (synthesized by Integrated DNA Technologies) (Supplementary Table 1). The signals of group-specific 16S rRNA gene were normalized with that of the total eubacteria 16S rRNA gene in each sample. The primers used are described in Supplementary Table 1.

       Assessment of C rodentium Load

      Figure thumbnail fx1
      Supplementary Figure 1Hypo-inflammatory responses after ethanol treatment in GPR41−/− and GPR43−/− mice. Histological changes in the distal colon after rectal administration of 50% ethanol. Colon tissues in paraffin were cut into 6-μm−thick sections and stained with H&E. The sections were graded on a 0−4 scale, as described in the . Significant differences (P < .05) from WT mice.
      Figure thumbnail fx2
      Supplementary Figure 2Defective cytokine and T-cell responses to TNBS in the gut of GPR41−/− and GPR43−/− mice. (A) Expression of signature genes for Th1, Th17, and regulatory T cells in the distal colon. (B) Frequencies of Th1, Th17, and regulatory T cells in the intestinal lamina propria and mesenteric lymph node (MLN). All of the data were obtained on day 3 after the TNBS treatment. Pooled data obtained from 5 independent experiments are shown. Significant differences (P < .05) from the untreated WT mice or TNBS-treated WT mice.∗∗ IFN-γ, interferon gamma.
      Figure thumbnail fx3
      Supplementary Figure 3GPR41−/− and GPR43−/− mice were less susceptible to TNBS-induced inflammation. Histological changes in the distal colon after rectal administration of TNBS in 50% ethanol. Colon tissues in paraffin were cut into 6-μm–thick sections and stained with H&E. The sections were graded on a 0–4 scale, as described in the . Significant differences (P < .05) from WT mice.
      Figure thumbnail fx4
      Supplementary Figure 4Reduced blood IL-6 concentration in GPR41−/− and GPR43−/− mice during C rodentium infection. Serum IL-6 level was examined in WT and KO mice 2 weeks after infection with C rodentium. Significant differences (P < .05) from WT mice.
      Figure thumbnail fx5
      Supplementary Figure 5Delayed induction of inflammatory cytokines and chemokines in GPR41−/− and GPR43−/− mice during C rodentium infection. (A) Expression of cytokines in the cecum (n = 13−15/group). (B) Expression of CXCL1 and CXCL2 in the cecum (n = 10−12/group). Quantitative real-time polymerase chain reaction was performed. Combined data of 3 independent experiments are shown. Significant differences (P < .05) from GPR41−/− () or GPR43−/− (∗∗) mice.
      Figure thumbnail fx6
      Supplementary Figure 6GPR41 and GPR43 do not affect gut permeability in the absence of immunological challenges. (A) Gut permeability and (B) expression of tight junction protein genes were examined by quantitative real-time polymerase chain reaction in unchallenged WT, GPR41−/−, and GPR43−/− mice. Pooled data obtained from 3 experiments are shown.
      Figure thumbnail fx7
      Supplementary Figure 7No significant changes in commensal bacteria in the gut of GPR41−/− or GPR43−/− mice. Levels of major bacterial groups in the gut were analyzed by quantitative polymerase chain reaction examination of the 16S rRNA gene isolated from the stool of WT, GPR41−/−, and GPR43−/− (n = 8−9 for each group) mice using the primers listed in . The copy numbers of 16S rRNA genes for specific bacterial groups were normalized by that of the total eubacteria. Abundance of the commensal bacterial groups is shown relative to WT levels.
      Figure thumbnail fx8
      Supplementary Figure 8Comparison of the roles of SCFA receptors expressed by non−bone marrow vs marrow-derived cells in mediating the inflammatory response to TNBS. (A) Weight change after TNBS treatment in marrow-transferred mice (n = 10−23/group). (B) Stool consistency scores (n = 10−15/group). (C) Gross appearance and length of the colon. (D) Infiltration of the colon by neutrophils. (E) Th1 frequency in the colon. All of the data were obtained on day 3 after TNBS treatment. Pooled data obtained from 3 independent experiments are shown. Significant differences (P < .05) from WT→WT mice or between indicated groups.∗∗
      Figure thumbnail fx9
      Supplementary Figure 9SCFA-dependent expression of IL-6 and chemokines by colonic ECs was suppressed by pertussis toxin. Expression of IL-6, CXCL1, and CXCL10 by ECs in response to SCFAs and bacterial products (LPS or commensal bacterial extract) was examined with quantitative real-time polymerase chain reaction. ECs, established for 3 days, were treated with pertussis toxin (PTX, 0.1 μg/mL; Toxin Technology, Sarasota, FL) for 1 h before treatment with the SCFAs for 6 days. Cells were then treated with LPS (1 μg/mL) or commensal bacterial extract (20 μg/mL) for 24 h. Pooled data from 3 independent experiments (n = 3) are shown. *Significant differences (P < .05) from the untreated WT control groups.
      Figure thumbnail fx10
      Supplementary Figure 10Effects of signaling molecule inhibitors on SCFA-dependent expression of neutrophil-attracting chemokines by epithelial cells. ECs, established for 3 days, were treated with inhibitors and SCFAs for 3 days. Commonly used concentrations of the inhibitors without significant off-target effects were used. Cells were then treated with LPS (1 μg/mL) for 24 h before quantitative real-time polymerase chain reaction analysis of CXCL1 (A) and CXCL2 (B). Pooled data obtained from 3 independent experiments (n = 3) are shown. Significant differences (P < .05) between control and inhibitor-treated groups for C2 or C3∗∗.
      Figure thumbnail fx11
      Supplementary Figure 11Proposed roles of SCFA signals in EC-mediated immune responses in the intestine. SCFAs are most abundant microbial metabolites in the gut and activate GPR41 and GPR43 expressed by colonic ECs. SCFA activation of ECs through GPR41 and GPR43 is required for timely induction of inflammatory cytokines and chemokines such as CXCL1, CXCL2, and CXCL10 to recruit leukocytes. The immune response enhanced by SCFAs increases gut permeability, and this process is delayed in GPR41- or GPR43-deficient mice. Based on the results described in this article, we propose that SCFA signals induce the following sequence of events: SCFAs→activation of GPR41/GPR43→activation of the ERK and p38 pathways→induction of inflammatory cytokines and chemokines→leukocyte infiltration and gut permeability change→increased immune responses (including T-cell response). Overall, the immune response enhanced by SCFAs is required for effective clearance of pathogens in the intestine. The same protective immune response can mediate tissue inflammation.
      Supplementary Table 1Primers Used in This Study
      IL17A-FGAC TCT CCA CCG CAATG
      IL17A-RCGG GTC TCT GTT TAG GCT
      RoRγt-FAGC CAC TGC ATT CCC AGT TTC T
      RoRγt-RTGA AAG CCG CTT GGC AAA CT
      IFNγ-FAGA CAA TCA GGC CAT CAG CA
      IFNγ-RCGA ATC AGC AGC GAC TCC TTT
      T-bet-FATG CCG CTG AAT TGG AAG GT
      T-bet-RACC TCG CAG AAA GCC ATG AAG T
      FoxP3-FCAG CTG CCT ACA GTG CCC CTA G
      FoxP3-RCAT TTG CCA GCA GTG GGT AG
      IL6-FCTG GTC TTC TGG AGT ACC ATA GC
      IL6-RTGC CGA GTA GAT CTC AAA GTG AC
      IL12-FCTA GGA TCG GAC CCT GCA GG
      IL12-RCTA GGA TCG GAC CCT GCA GG
      CCL2-FGTT GGC TCA GCC AGA TGC A
      CCL2-RAGC CTA CTC ATT GGG ATC ATC TTG
      CCL5-FTCA CCA TCA TCC TCA CTG C
      CCL5-RTGA ACC CAC TTC TTC TCT GG
      CXCL1-FCGT CGC TTC TCT GTG CA
      CXCL1-RTGG GGA GAC CTT TTA GCA
      CXCL2-FAAC CAC CGA GCT ACA GG
      CXCL2-RCCT TTC GAG GTC AGT TAG C
      CXCL10-FGGT CTG AGT GGG ACT CAA GG
      CXCL10-RTTT GGC TAA ACG CTT TCA TT
      CXCL13-FGCA GAA TGA GGC TCA GCA CAG
      CXCL13-RAGA GAA TGA GTG ACC TCG AAC
      MMP9-FTGT ACA CAG GCA AGA CCG TG
      MMP9-RCTC CAG CAT CCA GTG CAT CG
      MMP10-FCCT TCA GAC TTA GAT GCT GCC
      MMP10-RGTA CTA CTA GTC GTG TCG TCG
      IL1β-FGTA CAA GGA GAA CAA AGC AAC
      IL1β-RCTG TGC CTA AGG TAC CAC TTC
      EU338-FACT CCT ACG GGA GGC AGC AGT
      EU338-RATT ACC GCG GCT GCT GGC
      Enterobacteriaceae-Uni515FGTG CCA GCA GCC GCG GTA A
      Enterobacteriaceae-Ent826RGCC TCA AGG GCA CAA CCT CCA AG
      Bacteroides-BactF285GGT TCT GAG AGG AGG TCC C
      Bacteroides-UniR338GCT GCC TCC CGT AGG AGT
      Mouse intestinal bacteroides-Uni516FCCA GCA GCC GCG GTA ATA
      Mouse intestinal bacteroides-MIBR677GCG ATT CCG CAT ACT TCT C
      Clostridium coccoides-UniF338ACT CCT ACG GGA GGC AGC
      Clostridium coccoides-C.cocR491GCT TCT TAG TCA GGT ACC GTC AT
      Clostridium perfrigens-Cperf165FCGC ATA ACG TTG AAA GAT GG
      Clostridium perfrigens-Cperf269RCCT TGG TAG GCC GTT ACC C
      Segmented filamentous bacteria-SFB736FGAC GCT GAG GCA TGA GAG CAT
      Segmented filamentous bacteria-SFB844RGAC GGC ACG GAT TGT TAT TCA
      Bacillus-FGCG GCG TGC CTA ATA CAT GC
      Bacillus-RCTT CAT CAC TCA CGC GGC GT

      References

        • Ventura M.
        • Turroni F.
        • Canchaya C.
        • et al.
        Microbial diversity in the human intestine and novel insights from metagenomics.
        Front Biosci. 2009; 14: 3214-3221
        • Hamer H.M.
        • De Preter V.
        • Windey K.
        • et al.
        Functional analysis of colonic bacterial metabolism: relevant to health?.
        Am J Physiol Gastrointest Liver Physiol. 2012; 302: G1-G9
        • Jarchum I.
        • Pamer E.G.
        Regulation of innate and adaptive immunity by the commensal microbiota.
        Curr Opin Immunol. 2011; 23: 353-360
        • Sartor R.B.
        Key questions to guide a better understanding of host-commensal microbiota interactions in intestinal inflammation.
        Mucosal Immunol. 2011; 4: 127-132
        • Macia L.
        • Thorburn A.N.
        • Binge L.C.
        • et al.
        Microbial influences on epithelial integrity and immune function as a basis for inflammatory diseases.
        Immunol Rev. 2012; 245: 164-176
        • Le Poul E.
        • Loison C.
        • Struyf S.
        • et al.
        Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation.
        J Biol Chem. 2003; 278: 25481-25489
        • Brunkhorst B.A.
        • Kraus E.
        • Coppi M.
        • et al.
        Propionate induces polymorphonuclear leukocyte activation and inhibits formylmethionyl-leucyl-phenylalanine-stimulated activation.
        Infect Immun. 1992; 60: 2957-2968
        • Menzel T.
        • Luhrs H.
        • Zirlik S.
        • et al.
        Butyrate inhibits leukocyte adhesion to endothelial cells via modulation of VCAM-1.
        Inflamm Bowel Dis. 2004; 10: 122-128
        • Hague A.
        • Elder D.J.
        • Hicks D.J.
        • et al.
        Apoptosis in colorectal tumour cells: induction by the short chain fatty acids butyrate, propionate and acetate and by the bile salt deoxycholate.
        Int J Cancer. 1995; 60: 400-406
        • Kles K.A.
        • Chang E.B.
        Short-chain fatty acids impact on intestinal adaptation, inflammation, carcinoma, and failure.
        Gastroenterology. 2006; 130: S100-S105
        • Thwaites D.T.
        • Anderson C.M.
        H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine.
        Exp Physiol. 2007; 92: 603-619
        • Brown A.J.
        • Goldsworthy S.M.
        • Barnes A.A.
        • et al.
        The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids.
        J Biol Chem. 2003; 278: 11312-11319
        • Tazoe H.
        • Otomo Y.
        • Kaji I.
        • et al.
        Roles of short-chain fatty acids receptors, GPR41 and GPR43 on colonic functions.
        J Physiol Pharmacol. 2008; 59: 251-262
        • Xiong Y.
        • Miyamoto N.
        • Shibata K.
        • et al.
        Short-chain fatty acids stimulate leptin production in adipocytes through the G protein-coupled receptor GPR41.
        Proc Natl Acad Sci U S A. 2004; 101: 1045-1050
        • Samuel B.S.
        • Shaito A.
        • Motoike T.
        • et al.
        Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41.
        Proc Natl Acad Sci U S A. 2008; 105: 16767-16772
        • Maslowski K.M.
        • Vieira A.T.
        • Ng A.
        • et al.
        Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43.
        Nature. 2009; 461: 1282-1286
        • Sina C.
        • Gavrilova O.
        • Forster M.
        • et al.
        G protein-coupled receptor 43 is essential for neutrophil recruitment during intestinal inflammation.
        J Immunol. 2009; 183: 7514-7522
        • Wirtz S.
        • Neufert C.
        • Weigmann B.
        • et al.
        Chemically induced mouse models of intestinal inflammation.
        Nat Protoc. 2007; 2: 541-546
        • Simmons C.P.
        • Clare S.
        • Ghaem-Maghami M.
        • et al.
        Central role for B lymphocytes and CD4+ T cells in immunity to infection by the attaching and effacing pathogen Citrobacter rodentium.
        Infect Immun. 2003; 71: 5077-5086
        • Kang S.G.
        • Park J.
        • Cho J.Y.
        • et al.
        Complementary roles of retinoic acid and TGF-beta1 in coordinated expression of mucosal integrins by T  cells.
        Mucosal Immunol. 2011; 4: 66-82
        • Booth C.
        • O'Shea J.A.
        Isolation and culture of intestinal epithelial cells.
        in: Freshney R.I. Freshney M.G. Culture of Epithelial Cells. 2nd ed. Wiley-Liss, Inc., Hoboken, NJ2002: 303-335
        • Boirivant M.
        • Amendola A.
        • Butera A.
        • et al.
        A transient breach in the epithelial barrier leads to regulatory T-cell generation and resistance to experimental colitis.
        Gastroenterology. 2008; 135: 1612-1623 e5
        • Wells J.M.
        • Loonen L.M.
        • Karczewski J.M.
        The role of innate signaling in the homeostasis of tolerance and immunity in the intestine.
        Int J Med Microbiol. 2010; 300: 41-48
        • Wullaert A.
        Role of NF-kappaB activation in intestinal immune homeostasis.
        Int J Med Microbiol. 2010; 300: 49-56
        • Barnes M.J.
        • Powrie F.
        Regulatory T cells reinforce intestinal homeostasis.
        Immunity. 2009; 31: 401-411
        • Vinolo M.A.
        • Ferguson G.J.
        • Kulkarni S.
        • et al.
        SCFAs induce mouse neutrophil chemotaxis through the GPR43 receptor.
        PLoS One. 2011; 6: e21205
        • Yonezawa T.
        • Kobayashi Y.
        • Obara Y.
        Short-chain fatty acids induce acute phosphorylation of the p38 mitogen-activated protein kinase/heat shock protein 27 pathway via GPR43 in the MCF-7 human breast cancer cell line.
        Cell Signal. 2007; 19: 185-193
        • Lau E.
        • Ronai Z.A.
        ATF2—at the crossroad of nuclear and cytosolic functions.
        J Cell Sci. 2012; 125: 2815-2824
        • Sanz Y.
        • Nadal I.
        • Sanchez E.
        Probiotics as drugs against human gastrointestinal infections.
        Recent Pat Antiinfect Drug Discov. 2007; 2: 148-156
        • Milo L.A.
        • Reardon K.A.
        • Tappenden K.A.
        Effects of short-chain fatty acid-supplemented total parenteral nutrition on intestinal pro-inflammatory cytokine abundance.
        Dig Dis Sci. 2002; 47: 2049-2055
        • Saemann M.D.
        • Bohmig G.A.
        • Osterreicher C.H.
        • et al.
        Anti-inflammatory effects of sodium butyrate on human monocytes: potent inhibition of IL-12 and up-regulation of IL-10 production.
        FASEB J. 2000; 14: 2380-2382