Fig 1.
SCFAs bind to PYRIN domain–containing proteins.
(A) Schematic representation of the conjugation of propionate, acrylate, and butyrate to amino-modified affinity beads using succinate anhydride, malate anhydride, and glutaric anhydride, respectively. (B) Identification of ASC/PYCARD as a SCFA-binding protein. Propionate (Pro)-, acrylate (Acryl)-, or butyrate (But)-conjugated beads were incubated with U937 cell lysate. Bound proteins were visualized by silver staining and identified by ESI-MS peptide sequencing. (C) Competition assay for binding between ASC and propionate. Recombinant ASC was pretreated with propionate, butyrate, or lactate and then incubated with control (–) or propionate-conjugated beads. Bound protein was visualized by silver staining. (D) Alignment of the a.a. sequences of the PYRIN domains of ASC and NLRP1, 3, 4, and 6 using the Genetyx program. (E) Propionate binds to the Lys-rich domain of ASC/PYCARD. Recombinant ASC (full-length or the deletion mutants) was incubated with control (–) or propionate-conjugated beads, and bound proteins were visualized by silver staining. (F) Propionate-binding assays were performed with PYRIN domain–containing NLRPs (NLRPs 1, 3, 4, and 6) using propionate-conjugated beads. a.a., amino acid; ASC, apoptosis-associated speck-like protein; CARD, caspase activation and recruitment domain; ESI-MS, electrospray ionization–mass spectroscopy; Lys, lysine; NLRP, nucleotide-binding oligomerization domain-like receptor protein; SCFA, short-chain fatty acid.
Fig 2.
SCFAs induce inflammasome activation by enhancing oligomerization of the inflammasome complex.
(A) SCFAs promote the binding between ASC and NLRP3 in vitro. GST or GST-ASC was incubated with fluorescently labeled NLRP3 in the presence or absence of propionate (left), butyrate (middle), or lactate (right). Proteins were visualized by fluorescence imaging (top) or Coomassie Brilliant Blue staining (bottom). (B) Expression vector for FLAG-ASC and/or NLRP3 was transfected into HEK293T cells treated with propionate, butyrate, or lactate. Cell lysates were subjected to coimmunoprecipitation using anti-FLAG resin, and bound proteins were visualized by western blotting using anti-FLAG or anti-NLRP3 antibody. (C) U937 cells were incubated with LPS/ATP and treated with propionate (Pro), butyrate (But), or lactate (Lac). The IL-1β (left) and IL-18 (right) levels in the culture media were measured by ELISA. Data are the mean ± SD of three independent assays. *P < 0.05, **P < 0.01. Data are listed in S1 Data. (D) The effects of SCFA derivatives on IL-1β production were analyzed as shown above. C2, acetate; C3, propionate; C4, butyrate; C5, valerate; C6, caproate; C7, enanthate; C8, caprylate; Lac, lactate; MeP, 2-methylpropanoic acid; Ala, alanine; Mal, maleate; Suc, succinate; Cit, citrate. Data are the mean ± SD of three independent assays. *P < 0.05, **P < 0.01 versus LPS/ATP treatment. Data are listed in S1 Data. ASC, apoptosis-associated speck-like protein; GST, glutathione-S-transferase; IL, interleukin; IP, immunoprecipitation; LPS, lipopolysaccharide; NLRP, nucleotide-binding oligomerization domain-like receptor protein; SCFA, short-chain fatty acid; SD, standard deviation.
Fig 3.
SCFAs enhance the elimination of S. Typhimurium in macrophages by increasing inflammasome activity.
(A) BMDMs derived from wild-type, ASC−/−, or GPR43−/− mice were infected with S. Typhimurium strain A at multiplicity of infection of 5 for 10 minutes and then incubated in DMEM containing 100 μg/mL gentamycin for 15 hours with or without treatment with 10 mM acetate (Ace), propionate (Pro), butyrate (But), or lactate (Lac). The percentages of surviving S. Typhimurium strain A in comparison with untreated macrophages (ctrl) are shown. Data are the mean ± SD of three independent assays. *P < 0.05, **P < 0.01. Data are listed in S1 Data. (B) BMDMs were prepared as described above, and cell supernatants were collected and precipitated with 10% trichloroacetic acid. The precipitated proteins were subjected to western blotting with an anti-caspase-1 antibody. (C and D) BMDMs were prepared as described above, and cell supernatants were collected and LDH release and IL-1β production in the cell culture media as determined by LDH assay and ELISA, respectively. Data are the mean ± SD of three independent assays. *P < 0.05, **P < 0.01. Data listed in S1 Data. (E and F) BMDMs were treated with the MCT inhibitor SR13800 for 24 hours prior to S. Typhimurium strain A infection, and S. Typhimurium strain A–infected BMDMs were prepared as described above. The cells were lysed, and the bacterial cells within BMDMs were counted. IL-1β levels in the culture media were determined by ELISA. Data are the mean ± SD of three independent assays. *P < 0.05. Data listed in S1 Data. BMDM, bone marrow–derived macrophage; CFU, colony-forming unit; DMEM, Dulbecco’s modified Eagle’s medium; IL, interleukin; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; ND, not detected (below the detection limit); S. Typhimurium, S. enterica serovar Typhimurium; SCFA, short-chain fatty acid; SD, standard deviation.
Fig 4.
SCFAs enhance host defense against S. Typhimurium infection in an ASC-dependent manner.
(A) SPF wild-type mice were cohoused with SPF ASC−/− mice at a 1:1 gender ratio for 7 days and then were infected with S. Typhimurium strain A. Survival of SPF wild-type (●) and SPF ASC−/− mice (■) infected with S. Typhimurium strain A (left panel). Survival of antibiotic-treated wild-type (●) and ASC−/− mice (■) infected with S. Typhimurium strain A (right panel). P-values were determined by the log-rank test (n = 6 per group). Data are listed in S1 Data. (B) Treatment scheme followed to analyze the effect of SCFA administration on the susceptibility of antibiotic-treated mice to S. Typhimurium strain A. SPF mice were given antibiotics for 4 weeks. SCFAs were given 1 week before S. Typhimurium strain A infection. The administration of antibiotics was stopped before S. Typhimurium strain A infection. (C and D) Effects of propionate, butyrate, and lactate on the survival of wild-type (C) and ASC−/− (D) mice infected with S. Typhimurium strain A. The mice were given antibiotics for 1 month prior to being given drinking water containing 300 mM propionate, 300 mM butyrate, or 300 mM lactate for 1 week. Then, mice were inoculated with S. Typhimurium (108 bacteria) orally. P-values were determined by the log-rank test (n = 6 per group). Data are listed in S1 Data. ASC, apoptosis-associated speck-like protein; S. Typhimurium, S. enterica serovar Typhimurium; SCFA, short-chain fatty acid; SPF, specific pathogen-free.
Fig 5.
SCFAs recruit neutrophils to S. Typhimurium–infected foci through ASC-dependent inflammasome activation.
(A and B) HE staining of cecum tissues from SPF wild-type or ASC−/− mice and antibiotic-treated wild-type and ASC−/− mice infected with S. Typhimurium strain A. Scale bars = 10 μm. (C) Cecal mucosa was immunostained with an anti-F4/80 or anti-Gr-1 antibody after infection with S. Typhimurium strain A. Scale bars = 100 μm. (D) IL-1β production in the cecum tissues was measured by ELISA. Data are the mean ± SD. *P < 0.05. Data are listed in S1 Data. (E) Detection of the neutrophil marker Gr-1 protein by western blotting. Cecum tissues were collected, homogenized, and analyzed by western blotting using anti-Gr-1 and anti-actin antibodies. ASC, apoptosis-associated speck-like protein; HE, hematoxylin–eosin; IL, interleukin; S. Typhimurium, S. enterica serovar Typhimurium; SCFA, short-chain fatty acid; SD, standard deviation; SPF, specific pathogen-free.
Fig 6.
SCFAs exert host-protective effects in a macrophage-dependent manner.
Mice were given antibiotics for 1 month prior to being given drinking water containing 300 mM propionate or 300 mM butyrate for 1 week. Then, clodronate liposomes (56 mg per kg of body weight) were intraperitoneally administrated to the mice 24 hours prior to oral infection with S. Typhimurium (108 bacteria). (A) Effect of clodronate liposomes (macrophage-depleting agent) on the survival of propionate- or butyrate-administrated wild-type mice infected with S. Typhimurium strain A. P-values were determined by the log-rank test (n = 6 per group). Data are listed in S1 Data. (B) Bacterial counts in cecum tissues, MLN, liver, and spleen were determined 2 days after infection. Cecum contents and each tissue were homogenized in PBS, and then the homogenates were plated on the Salmonella Shigella selection agar (Difco SS agar) and CFU were counted. Bars indicate the mean (n = 6 per group). *P < 0.05, **P < 0.01. Data are listed in S1 Data. (C) Immunostaining of cecum tissues after infection with S. Typhimurium strain A using anti-F4/80 (macrophage marker) and anti-Gr-1 (neutrophil marker) antibodies. Scale bars = 100 μm. (D) IL-1β production in the cecum as measured by ELISA. Data are the mean ± SD. *P < 0.05, ** P < 0.01. Data are listed in S1 Data. CFU, colony-forming unit; DAPI, 4',6-diamidino-2-phenylindole; IL, interleukin; MLN, mesenteric lymph node; S. Typhimurium, S. enterica serovar Typhimurium; SCFA, short-chain fatty acid; SD, standard deviation.
Fig 7.
Dietary fiber enhances host defense against S. Typhimurium infection.
(A) Dietary fiber (PHGG-R [mean molecular mass of 20 kDa] or PHGG-HG [mean molecular mass of 14 kDa]) was administered to SPF wild-type mice. Butyrate concentration in the cecal lumen as determined by LC-MS. Data are the mean ± SD (n = 5 per group). **P < 0.01. Data are listed in S1 Data. (B) Effect of each dietary fiber on the survival of wild-type infected with S. Typhimurium strain A (n = 6 per group). P-values were determined by the log-rank test. Data are listed in S1 Data. (C) Effect of each dietary fiber on the survival of ASC−/− mice infected with S. Typhimurium strain A (n = 5 per group). Data are listed in S1 Data. (D) IL-1β production in the cecum as determined by ELISA. Data are the mean ± SD. *P < 0.05. Data are listed in S1 Data. (E) Imaging mass spectrometric analysis of the localization of citrate, propionate, and butyrate/isobutyrate in cecum tissues from SPF wild-type mice (left panel), S. Typhimurium strain A–infected SPF wild-type mice (middle panel), and PHGGR-administrated S. Typhimurium strain A–infected SPF wild-type mice (right panel). Each tissue section was stained with HE (upper panel). Black dotted lines in imaging mass spectrometric images indicate a boundary between cecal lumen and cecal mucosa. Scale bars = 250 μm. ASC, apoptosis-associated speck-like protein; HE, hematoxylin–eosin; IL, interleukin; LC-MS, liquid chromatography–mass spectroscopy; PHGG, partially hydrolyzed guar gum; S. Typhimurium, S. enterica serovar Typhimurium; SD, standard deviation; SPF, specific pathogen-free.
Fig 8.
Schematic diagram of SCFA-induced inflammasome activation and defense against S. Typhimurium.
SCFAs, which are abundantly produced by fermentation of dietary fibers in the gut, induce inflammasome activation in infected macrophages by enhancing oligomerization of the inflammasome complex. Inflammasome activation contributes to the elimination of intracellular S. Typhimurium by pyroptosis. In addition, released pathogens are neutralized by infiltrating neutrophils, which are recruited to the inflamed tissue by IL-1β. ASC, apoptosis-associated speck-like protein; CARD, caspase activation and recruitment domain; IL, interleukin; LRR, leucine-rich repeat; NLRP, nucleotide-binding oligomerization domain-like receptor protein; PHGG, partially hydrolyzed guar gum; S. Typhimurium, S. enterica serovar Typhimurium; SCFA, short-chain fatty acid.