Fig 2: Caspase-1 and NLRP3 in THP-1 cells are involved in T. gondii-mediated IDO1 inhibition. (A and B) WT, CASP1-KO, NLRP1-KO, or NLRP3-KO THP-1 cells were left uninfected or infected with Pru T. gondii. (A) Levels of IL-1ß released into the culture supernatant were measured by ELISA. (B) The infected THP-1 cells were cocultured with Huh7 cells and then left untreated or treated with IFN-?. The parasite survival rates were measured by luciferase assay. Indicated values represent means ± SD (three biological replicates per group from three independent experiments) (panels A and B). ***, P < 0.001; **, P < 0.01; N.S., not significant (Student's t test).

Fig 3: The distribution of NLRP1, NLRP2, NLRP3, NLRP6, NLRP12, and absent in melanoma 2 (AIM2) in the gingival epithelium and lamina propria of healthy and periodontitis patients. Full-thickness gingival tissue specimens including both the epithelium and connective tissue were obtained from healthy subjects (n = 5) and patients with severe chronic periodontitis (n = 5). Samples were fixed in 10% formalin for immunohistochemistry assays to detect the expression of NLRP1, NLRP2, NLRP3, NLRP6, NLRP12, and AIM2 (P: periodontitis specimens, n = 5; H: healthy gingiva, n = 5; arrows: positive stain; and magnification: 400×). *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 4: Increased expression of inflammasome components in thyroid cells on stimulation with IFN-? and tumor necrosis factor-a (TNF-a). (A) Changes in NLRP3, absent in melanoma 2 (AIM2), NLRP1, NLRC4, ASC, and caspase-1 protein expression detected by western blot after incubation with 500 IU/ml IFN-? or 500 IU/ml TNF-a for 24 h. Changes in NLRP3, AIM2, NLRP1, NLRC4, ASC, Caspase-1, IL-1ß, and IL-18 mRNA expression detected by real-time PCR after incubation with (B) gradient IFN-? (250, 500, and 1,000 IU/ml) for 24 h or (C) gradient TNF-a (125, 250, and 500 IU/ml) for 24 h. The relative mRNA and protein expression levels were corrected to those of untreated controls. (D) IL-18 and IL-1ß levels in cell supernatant, as determined by enzyme-linked immunosorbent assay, after stimulation with 500 IU/ml IFN-?, 500 IU/ml TNF-a or a combination of the two for 24 h. The columns represent the mean ± SD values. Data were obtained from three independent experiments. One-way ANOVA followed by Bonferroni correction was used for paired comparisons. *P < 0.05, **P < 0.01.

Fig 5: IL-1ß production and signaling are essential for GRA15-mediated IDO1 inhibition. (A) WT or MyD88-KO Huh7 cells were left untreated or treated with the indicated cytokines. Expression of IDO1 in the cell lysates was detected by Western blotting. (B and C) WT, Atg16L1-KO, or IDO1-KO Huh7 cells were left untreated or treated with IFN-? for 24 h and then infected with Pru T. gondii. (B) The parasite survival rates were measured by luciferase assay. (C) The parasite number per vacuole after 24 h postinfection was measured by indirect immunofluorescent assay (IFA). (D) WT or IDO1-KO Huh7 cells were left untreated or treated with the indicated cytokines for 24 h and then infected with Pru T. gondii. The parasite survival rates were measured by luciferase assay. (E) THP-1 cells were infected with WT or GRA15-KO Pru T. gondii. The infected THP-1 cells were cocultured with Huh7 cells and then left untreated or treated with IFN-?. Expression of IDO1 in the cell lysates was detected by Western blotting. (F) THP-1 cells were infected with GRA15-KO Pru T. gondii. The infected THP-1 cells were cocultured with WT or IDO1-KO Huh7 cells and then left untreated or treated with IFN-?. The parasite survival rates were measured by luciferase assay. (G) THP-1 cells were infected with Pru T. gondii. The infected THP-1 cells were cocultured with WT or MyD88-KO Huh7 cells and then left untreated or treated with IFN-?. Expression of IDO1 in cell lysates was detected by Western blotting. (H) WT, CASP1-KO, NLRP1-KO, or NLRP3-KO THP-1 cells were left uninfected or infected with Pru T. gondii. The infected THP-1 cells were cocultured with Huh7 cells and then left untreated or treated with IFN-?. Expression of IDO1 in the cell lysates was detected by Western blotting. (I) WT or Irgm1/3 double-knockout (DKO) primary mouse hepatocytes were left untreated or treated with the indicated cytokines for 24 h and then infected with Pru T. gondii. The parasite survival rates were measured by luciferase assay. (J) Primary human hepatocyte or primary mouse hepatocytes were left untreated or treated with the indicated cytokines. Expression of indicated proteins in cell lysates was detected by Western blotting. (K) WT or IRGM-KO Huh7 cells were left untreated or treated with the indicated cytokines for 24 h and then infected with wild-type Pru T. gondii. The parasite survival rates were measured by luciferase assay. (L) WT or IRGM-KO Huh7 cells were left untreated or treated with the indicated cytokines. Expression of indicated proteins in cell lysates was detected by Western blotting. Each Western blot image is representative of three independent experiments (A, E, G, H, J, and L). Indicated values represent means ± SD (three biological replicates per group from three independent experiments) (B to D, F, I, and K). **, P < 0.01; N.S., not significant (Student's t test).