Fig 2: Inhibiting IDO-1 with 1-MT or INCB24360 reduced DTR-dextran uptake by RAW264.7 and BV-2 cells induced by IFN-?.(A, B) The typical images of engulfed DTR-dextran in IFN-? induced RAW264.7 (A) or BV-2 (B) cells after administration of dextran for 1 h in the control, IFN-?, IFN-?+1-MT and IFN-? + INCB24360 groups. The phagocytic particles larger than 0.75 µm were analyzed by IMARIS 9.6. Scale bars, 40 µm. (C, D) Quantification of DTR-dextran particles in RAW264.7 cells (C) and BV-2 cells (D). Single-cell area X Dextran average grayscale was used as statistical data; n = 15. (E, F) DTR-dextran particles larger than 0.75 µm in the control, IFN-?, IFN-?+1-MT and IFN-? + INCB24360 groups of IFN-? induced RAW264.7 cells (E) or BV2 cells (F). n = 15. (G) Phagocytosis of latex beads in BV-2 cells treated with IFN-?, IFN-?+1-MT or IFN-? + INCB24360 for 24 h. The three statistical charts represent that the phagocytic ratio, the number of beads in each cell, the number of cells with one, two, three, or more beads. Scale bars, 100 µm. One-way ANOVA; all data are expressed as the means ± SEM. *, P<0.05, **, P<0.01; ns, no significant difference.

Fig 3: Other RIPK1 inhibitors and IDO inhibitor did not prevent the decrease in cell viability induced by sulfasalazine. (A) Huh7 and SK-HEP-1 cells were treated with vehicle (circle) or 20 µM necrostatin-1s (RIPK1 inhibitor, triangle), 20 µM GSK2982772 (RIPK1 inhibitor, inverted triangle), or 500 µM 1-methyl-D-tryptophan (IDO inhibitor, square) for 1 h, followed by sulfasalazine in a dose-dependent manner for 24 h. Cell viability was measured with a CellTiter-Glo® assay. (B) Cells were treated with 500 µM sulfasalazine for indicated periods and subjected to Western blot analysis. HT29 cells treated with vehicle or TSZ (50 ng/ml TNFa, 1 µM SM164 was 20 µM Z-VAD-FMK) for 12 h as a positive necroptosis model. Relative expression was calculated in compared with vehicle (8 h) group for IDO or HT29 (vehicle) group for others. Each experiment was repeated 3 times in triplicate and data are expressed as means ± SDs. N.S., not significant.

Fig 4: Inhibiting IDO-1 with 1-MT or INCB24360 reduced NLRP3 expression in RAW264.7 cells.(A) NLRP3, IDO-1 and IBA-1 triple-staining in the meninges of a human noncancerous brain (Yellow arrow, vessels; VL, vascular lumen). (B) The changes of NLRP3 and caspase-1 in RAW264.7 cells treated with IFN-?, 1-MT or INCB24360 for 24 h. The relative intensity of NLRP3 and caspase-1 in RAW264.7 cells as measured by ImageJ software. (C) The immunostained results of NLRP3 and iNOS in RAW264.7 cells treated with IFN-?, 1-MT or INCB24360 for 24 h. The relative NLRP3 or iNOS intensity in RAW264.7 cells as measured by ImageJ software. n=10. Scale bars, 80 µm. (D) The changes of NLRP3 and caspase-1 in RAW264.7 cells treated with IFN-?, IFN-?+1-MT or IFN-? + INCB24360 for 24 h. The relative intensity of NLRP3, caspase-1 in RAW264.7 cells as measured by ImageJ software. (E) The transcription levels of NLRP3 and caspase-1 in RAW264.7 cells after treatment with IFN-?, IFN-?+1-MT or IFN-? + INCB24360 for 24 h. (F) The immunostained images of NLRP3 and iNOS in RAW264.7 cells treated with IFN-?, IFN-?+1-MT or IFN-? + INCB24360 for 24 h. The relative NLRP3 or iNOS intensity in RAW264.7 cell as measured by ImageJ software. Scale bars, 80 µm. One-way ANOVA; all data are expressed as the means ± SEM. *, P<0.05, **, P<0.01; ns, no significant difference. Scale bars, 80 µm.

Fig 5: Inhibiting IDO-1 with INCB24360 or 1-MT suppresses IFN-? induced iNOS and TNFa increases in RAW264.7 cells.(A) iNOS, CD206 and IDO-1 expression in RAW264.7 cells treated with IFN-?, IFN-?+1-MT or IFN-? + INCB24360 for 24 h (12 h with IFN-?, following another 12 hours with 1-MT or INCB24360) and the relative intensity of iNOS, CD206, and IDO-1 in the RAW264.7 cells as measured by ImageJ. (B) The transcription levels of iNOS, TNFa, CD206 and Arg1 in RAW264.7 cells treated with IFN-?, IFN-?+1-MT or IFN-? + INCB24360 for 24 h. (C) The immunostained images of iNOS and CD206 in RAW264.7 cells treated by IFN-?, IFN-?+1-MT or IFN-? + INCB24360 for 24 h. The quantification of iNOS and CD206 intensity in RAW264.7 cells as measured by ImageJ software. Scale bars, 50 µm. One-way ANOVA; all data are expressed as the means ± SEM. *, P<0.05, **, P<0.01; ns, no significant difference.