Fig 2: Butyrate Supplementation Increases the Availability of AhR Ligands(A) Mean clinical score of control and butyrate-supplemented ABX-treated or untreated mice; y axis shows percentage swelling in antigen-injected knee compared to control knee (cumulative n = 8 per group, one representative experiment of two experiments is shown).(B) Bar chart shows expression of Il10 relative to ß-actin in splenic B cells isolated from ABX-treated WT or untreated mice (cumulative n = 3 per group).(C) Bar chart shows relative abundance of bacterial phyla in the stool of naive, control arthritic, or butyrate-supplemented arthritic mice (n = 4 per group).(D) XY graph shows operational taxonomic units (OTUs) of bacterial genera in butyrate-supplemented and control arthritic mice (n = 4 per group).(E–G) Bar charts shows levels of tryptophan, tryptamine, indole (E), L-Kynurenine, Kynurenic Acid (KYNA) (F), and 5-HIAA (G) in the stool of control arthritic WT and butyrate-supplemented arthritic mice (cumulative n = 5 per group).Data represent mean ± SE (A, two-way ANOVA; B, one-way ANOVA; E–G, Student’s t test). See also Figure S7.

Fig 3: 5-Hydroxyindole-3-Acetic Acid Increases Il10 Transcription by B cells In Vivo and In Vitro by Acting as a Ligand for AhR(A) Relative expression of Cyp1a1 and Il10 in total splenic B cells following 6-h culture with 5-HIAA or kynurenic acid (KYNA) compared to vehicle alone (n = 3, one of two experiments is shown).(B) Mean clinical score of control, 5-HIAA-gavaged, or KYNA-gavaged mice; y axis shows percentage swelling in antigen-injected knee compared to control knee (cumulative n = 8 per group, one representative experiment of two experiments is shown).(C) Bar charts show expression of Cyp1a1 and Il10 relative to ß-actin in splenic B cells isolated from control, 5-HIAA-gavaged, or KYNA-gavaged mice.(D) Mean clinical score of control or 5-HIAA-gavaged Mb1cre/+ mice or Ahrfl/-Mb1cre/+ mice; y axis shows percentage swelling in antigen-injected knee compared to control knee (cumulative n = 8 per group, one representative experiment of two experiments is shown).(E) Mean clinical score of control and butyrate-supplemented L-para-chlorophenylalanine (PCPA)-treated (tryptophanase inhibitor, TPH) or vehicle-treated mice; y axis shows percentage swelling in antigen-injected knee compared to control knee (cumulative n = 10 per group, one representative experiment of two experiments is shown).Data represent mean ± SE (A, Student’s t test; B, two-way ANOVA; C, one-way ANOVA; D and E, two-way ANOVA).

Fig 4: Adoptive transfer of MDSCs ameliorated radiation-induced intestinal injury and inflammation.Adoptive transfers of 0.5 Gy- or 5 Gy-induced splenic MDSCs were intravenously injected in mice on days 1 and 4 after irradiation. A Mice were injected with CFSE-labeled 0.5 Gy- or 5 Gy-induced splenic MDSCs (5 × 106 cells/100 µL) twice after irradiation. Fluorescence images of intestines were obtained 24 h after injections using an IVIS 200 image system. B Photomicrographs of colon sections and histological grades of mice 21 days after irradiation. Scale bar = 100 µm. C Photomicrograph of colons and measurements of colon length, survival rates, and body weights in mice 21 days after irradiation. D The relative expression of cytokines (IL10, TGF ß, IL6, IFN?, IL1ß, and TNFa) in the intestines of irradiated mice were measured using qRT-PCR. The data are represented as the mean ± SD (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001 vs. PBS. IR irradiation.

Fig 5: MDSCs and IL10 are increased in 0.5Gy-exposed mice.Mice were exposed to either 0.5 Gy or 5 Gy total body irradiation and analyzed seven days later. Representative dot plots of the gating strategies. A The population of CD11b+ /GR1+ cells. B gMDSCs (CD11b+ , Ly6G+, and Ly6Clow) and mMDSCs (CD11b+ , Ly6G-, and Ly6Chigh) from the spleens and intestines of irradiated mice were identified and analyzed by flow cytometry. C Representative dot plots of the gating strategy used to identify IL10+ cells. IL10+ cells were gated and the proportions of CD11b+ /GR1+ cells. These cells were gated on gMDSCs (CD11b+ , Ly6G+, and Ly6Clow) and mMDSCs (CD11b+ , Ly6G-, and Ly6Chigh) cells were evaluated from mouse spleens and intestines. D Representative immunofluorescence images of cells from spleen stained for GR1 (green), IL10 (red), and GR1/IL10 (yellow). DAPI staining (blue) was used to determine the number of nuclei. Scale bar = 100 µm. The data are represented as mean ± SD (n = 8). **P < 0.01, ***P < 0.001 vs. control.