Fig 2: iNOS expression in Huh7 cells is required for GRA15-mediated IDO1 inhibition. (A) Quantitative RT-PCR analysis of iNOS mRNA level in WT or MyD88-KO Huh7 cells that were left untreated or treated with the indicated cytokines was performed. (B) WT or iNOS-KO Huh7 cells were left untreated or treated with the indicated cytokines. Expression of indicated proteins in the cell lysates was detected by Western blotting. (C) WT, iNOS-KO, 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. (D to F) THP-1 cells were infected with Pru T. gondii. The infected THP-1 cells were cocultured with WT or iNOS-KO Huh7 cells and then left untreated or treated with IFN-?. (D) Levels of NO2 released into the culture supernatant were measured by ELISA. (E) Expression of indicated proteins in the cell lysates was detected by Western blotting. (F) The parasite survival rates were measured by luciferase assay. (G to K) WT or CASP1-KO (G) THP-1 cells were infected with WT or GRA15-KO Pru T. gondii (H) or with GRA15-KO parasites stably expressing empty or GRA15 expression vectors (K). The infected THP-1 cells were cocultured with WT, MyD88-KO (I), or IL-1R1-KO (J) Huh7 cells and then left untreated or treated with IFN-?. Expression of indicated proteins in the cell lysates was detected by Western blotting. Each Western blot image is representative of three independent experiments (B, E, and G to K). Indicated values represent means ± SD (three biological replicates per group from three independent experiments) (A, C, D, and F). **, P < 0.01; N.S., not significant (Student's t test).

Fig 3: AVL ameliorated ethanol-induced hepatic inflammation. (A) The effects of AVL on the protein expressions of NLRP3, IL6, F4/80, caspase1, IL1R1, IL1ß, ASC, and MPO. Densitometric values were normalized against GAPDH. The same GAPDH was used in Figure 2A,B, Figure 3D, and Panel 4A. (B) Representative QPCR analysis for NLRP3, ASC, IL6, caspase1-p10, IL1R1, and mature-IL1ß. (C) Immunohistochemical staining analysis of MPO (×200 and ×400 magnification), and immunofluorescence staining analysis of NLRP3 and F4/80 (×600 magnification). Data are presented as mean ± SD (n = 3). ### p < 0.001 compared with normal group; *p < 0.05, **p < 0.01, ***p < 0.001 compared with EtOH group; ns, not significant.

Fig 4: Low molecular weight of IL1R1/CD121a is specifically expressed in synMSCs which is majorly existed in the cytoplasmic region. (A)IL1R1/CD121a mRNA is expressed in synMSCs. Relative expression levels were analyzed by Q-PCR. Data are presented as mean ± SD (n = 3). (B) A short isoform of IL1R1/CD121a protein was detected in synMSCs (Western blot). To reconfirm the short isoform of IL1R1/CD121a in synMSCs, cells from 6 patients were subjected for the analyses and obtained the similar results (data not shown). See Supplemental figure 2 for uncropped images. (C) IL1R1/CD121a (in green) was co-stained with Na+/K + ATPase (in red) which was selected as marker of the plasma membrane. Cell nuclei was stained by DAPI (n = 4). (D) Pearson's correlation coefficient and Mander's overlap coefficient were calculated by the coloc2 plugin in Image J, and each point represents one region of interest. Selected regions of interest included over 100 discernible cells in total for each cell line [18, 19, 20]. Data are presented as mean ± SD (n = 4). **** indicates p < 0.001 by One-way ANOVA followed by the Tukey–Kramer method.

Fig 5: The growth-promoting effect of IL1ß on synMSCs is through type I IL1ß receptor, IL1R1/CD121a, although it is not presented on the cell membrane. (A) Population of the cells positive for IL1R1/CD121a measured by flow cytometer. Cells were maintained in the normal growth medium for 7days and stained with anti-CD121a antibody. Data are presented as mean ± SD (n = 5). (B) IL1ß (10 ng/mL) enhances MSC proliferation without affecting the IL1R1/CD121a-positive population. IL1R1/CD121a-positive cell population was measured by a flowcytometer (Left). Cell number was counted by hemocytometer (Right). Data are presented as mean ± SD (n = 4). (C) MTT assay indicated that IL1Ra (60 ng/mL) completely inhibited the growth-promoting effects of IL1ß (10 ng/mL) in synMSCs. Data are presented as mean ± SD (n = 6). **** indicates p < 0.001 by One-way ANOVA followed by the Tukey–Kramer method.