Journal: Mucosal immunology

Article Title: Tryptophan Metabolite Activation of the Aryl Hydrocarbon Receptor Regulates IL10 Receptor Expression on Intestinal Epithelia

doi: 10.1038/mi.2016.133

Figure Lengend Snippet: (A) qPCR of il10r1 and cyp1a1 transcript levels in T84 IEC in response to AHR ligands. Confluent monolayers of T84 cells were treated with Kyn (100 μM), FICZ (250 nM), or TCDD (30 nM) for 6 h. Error bars represent the S.E.M. of five replicates. (B) Luciferase-based assay of IL10R1 promoter activity in response to AHR ligands FICZ and Kyn. IFN- γ served as a positive control. Caco-2 IECs were transfected with pGL3 containing the IL10R1 promoter region or empty pGL3 vector as control, treated for 12h with 100 μM Kyn, 250 μM FICZ, or 10 ng/mL IFN- γ and luciferase activity measured, n=3 (***p<0.001). (C) Cell surface ELISA of IL10R1 expression in shControl, shRNA ARNT knockdown, and AHR inhibitor treated Caco-2 IEC. Confluent monolayers of Caco-2 IEC were treated with 100 μM Kyn, 250 nM FICZ, or 30 nM TCDD for 24h. Error bars represent the S.E.M. of triplicate samples, *p<0.05, **p<0.01. (D) Western blot analysis of IL10R1 levels in shRNA AHR knockdown Caco-2 IEC. Confluent monolayers of Caco-2 cells containing a non-template control (shNTC) or shRNA specific for AHR were treated with FICZ or Kyn for 24 h. (E) Densitometry of the western blot analysis in . Relative density of the IL10R1 bands is normalized to β-actin. (F) Western blot analysis of IL10R1 levels in shRNA ARNT knockdown T84 IEC. Confluent monolayers of T84 cells containing a non-template control (shNTC) or shRNA specific for ARNT were treated with Kyn, FICZ, or TCDD for 24 h. (G) Densitometry of the western blot analysis in . Relative density of the IL10R1 bands is normalized to β-actin.

Article Snippet: Where indicated, cells were treated with the AHR inhibitor CH223191 (Sigma, 10 μM), the IDO1 inhibitor 1-methyl-DL-tryptophan (1-MT, Sigma), recombinant human IFN- γ (10 ng/mL, R&D), or the AHR agonists FICZ (6-Formylindolo(3,2-b)carbazole; 100 nM – 1 μM; Enzo), Kyn (Kynurenine, Sigma, 100 μM), or TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, Sigma, 30 nM) in serum free media.

Techniques: Luciferase, Activity Assay, Positive Control, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Expressing, shRNA, Western Blot

Journal: Frontiers in Immunology

Article Title: Prostaglanin-E2 Potentiates the Suppressive Functions of Human Mononuclear Myeloid-Derived Suppressor Cells and Increases Their Capacity to Expand IL-10-Producing Regulatory T Cell Subsets

doi: 10.3389/fimmu.2019.00475

Figure Lengend Snippet: The capacity of LPS-IFN-γ stimulated M-MDSC to induce FoxP3 + Treg. (A) A representative analysis of FoxP3 + Treg is shown from the experiments in which M-MDSC (2 × 10 3 /well) were co-cultivated with allogeneic T cells for 3 days, followed by IL-2 treatment for the next 3 days. The presented histograms of FoxP3 and TGF-β are shown from CD4 + CD25 hi gates, and the markers were set according to FMO control. (B) The surface expression of PD-L1 and intracellular expression of IDO-1 were determined by flow cytometry after the staining of LPS/IFN-γ stimulated M-MDSC, and the results are shown as mean MFI or % ± SD of 3 independent experiments. * p < 0.05 paired T -test. (C) The summarized data are shown on the % of CD4 + CD25 hi FoxP3 + cells ± SD ( n = 3) induced in the co-cultures with M-MDSC that were carried out as in (A) , either in the presence or absence of 1-MT. * p < 0.05 as indicated by line (RM ANOVA, Tukey post-test).

Article Snippet: To assess the mechanisms of Treg induction, some M-MDSC/T cell co-cultures were supplemented with IDO-1 inhibitor 1-methyl-tryptophan (1-MT, 0.3 mM; Sigma-Aldrich Co.), blocking anti-ILT-3 or anti-ILT-4 Ab (both at 2 μg/mL; R&D Systems), or isotype control Ab (anti-rat IgG2b; Thermo Fisher Scientific).

Techniques: Expressing, Flow Cytometry, Staining

Journal: Free radical biology & medicine

Article Title: Indoleamine 2, 3-dioxygenase 1 aggravates acetaminophen-induced acute liver failure by triggering excess nitroxidative stress and iron accumulation.

doi: 10.1016/j.freeradbiomed.2021.07.008

Figure Lengend Snippet: Fig. 1. APAP-induced acute liver failure involved lipid peroxidation, excess nitrative stress and iron up-regulation, accompanied by increased expression of IDO1. (A) Representative images of surface morphology and color of mice livers in different groups. (B) Hematoxylin-eosin (H&E) staining and TUNEL staining of liver paraffin sections were shown from control group and 300 mg/kg APAP treated group (dotted lines in white showed damaged areas). (C) Serum concentrations of AST/ALT between control group and APAP group. (D) The RNS production after acute liver failure stained positive for NP3 probe (blue). PI nucleic counterstain appeared in red. (E)TGF-βr and IFN-γ protein expression. GAPDH served as a loading control respectively. (F-H) The hepatic levels of SOD activity, GSH and MDA in mice of control group and APAP group were determined.(I)Iron concentration (including total iron, ferrous iron and ferric iron) in control group and APAP-treated mice was detected. (J) Representative electron microscope images of morphological structure of mitochondria in hepatocytes (arrows in white showed lesions). (K) Western blot analysis reveals increased TfR and Tf after 24 h of APAP injection in mice. GAPDH served as a loading control respectively. (L) Western blot and immunohistochemical representative images of IDO1 in control group and APAP group. GAPDH served as a loading control respectively. The density of protein, the fluorescence intensity and the area of immunohistochemistry staining was measured using image J software. Statistical analysis demonstrated the mean + SEM, *p<0.05, **p<0.01, ***p<0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The IDO1 inhibitor 1-MT (1-methyl-tryptophan, Selleck) was solubled in dimethyl sulfoxide (DMSO) and diluted to 50 μM with filtered egg water.

Techniques: Expressing, Staining, TUNEL Assay, Control, Activity Assay, Concentration Assay, Microscopy, Western Blot, Injection, Immunohistochemical staining, Fluorescence, Immunohistochemistry, Software

Journal: Free radical biology & medicine

Article Title: Indoleamine 2, 3-dioxygenase 1 aggravates acetaminophen-induced acute liver failure by triggering excess nitroxidative stress and iron accumulation.

doi: 10.1016/j.freeradbiomed.2021.07.008

Figure Lengend Snippet: Fig. 3. IDO1 deficiency enhanced antioxidant capacity and suppressed lipid peroxidation in ALF. (A) Serum concentrations of AST and ALT were measured. (B, C) Hematoxylin–eosin (H&E) stained and TUNEL stained sections of the livers were shown (dotted lines in white showed damaged areas). (D) Western blot analysis relative density ratios of iNOS, COX-2 and Cyto-C expression in different groups of WT and IDO1−/−mice livers at 24 h after APAP administration. GAPDH served as a loading control. (E) Immunohistochemical representative images of 4-HNE in different groups. (F, G) MDA and GSH levels in mice of different indicated groups were determined. (H) Representative ROS probe detection pictures in LO2 cells were shown. Quantitative analysis in above panel. The density of protein and the fluorescence intensity were measured using image J software. Statistical analysis demonstrated the mean + SEM, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The IDO1 inhibitor 1-MT (1-methyl-tryptophan, Selleck) was solubled in dimethyl sulfoxide (DMSO) and diluted to 50 μM with filtered egg water.

Techniques: Staining, TUNEL Assay, Western Blot, Expressing, Control, Immunohistochemical staining, Fluorescence, Software

Journal: Free radical biology & medicine

Article Title: Indoleamine 2, 3-dioxygenase 1 aggravates acetaminophen-induced acute liver failure by triggering excess nitroxidative stress and iron accumulation.

doi: 10.1016/j.freeradbiomed.2021.07.008

Figure Lengend Snippet: Fig. 4. IDO1 deficiency performed stronger resistance to excess nitrative stress. (A) Immunofluorescence staining of 3-NT (red) in livers in vehicle, APAP- treated, IDO1−/−vehicle and IDO1−/−APAP treated mice. (B) Fluorescence micrographs of ONOO- probe detection (blue) of livers. Nuclei were stained with PI (red). The density of protein and the fluorescence intensity were measured using image J software. Statistical analysis demonstrated the mean + SEM, *p<0.05, **p<0.01, ***p<0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The IDO1 inhibitor 1-MT (1-methyl-tryptophan, Selleck) was solubled in dimethyl sulfoxide (DMSO) and diluted to 50 μM with filtered egg water.

Techniques: Immunofluorescence, Staining, Fluorescence, Software

Journal: Free radical biology & medicine

Article Title: Indoleamine 2, 3-dioxygenase 1 aggravates acetaminophen-induced acute liver failure by triggering excess nitroxidative stress and iron accumulation.

doi: 10.1016/j.freeradbiomed.2021.07.008

Figure Lengend Snippet: Fig. 5. Inhibition of IDO1 contributed to hepatocytes production and inflammatory cells reduction. (A) Immunofluorescence staining of F4/80 in livers. (B, C) The serum levels of IL-6 and TGF-β in mice of different indicated groups were detected. (D) Schematic illustration of the zebrafish drug toxicity experiment. Zebrafish were cultured for 48 h after exposed to egg water or medias for 48 h before imaging respectively. (E) Survival proportions of zebrafish from 0 to 48 h post exposure to egg water, or APAP (5 mM, 10 mM). Zebrafish survival status were observed at 0, 12, 24, 36 and 48 hpe, n=30 per group. (F) Quantitative analysis of hepatocytes fluorescence in zebrafish. Figures are magnified as 64 × . n=8-10 per group. (G, H) Quantitative analysis of systemic neutrophils and macrophages fluorescence in zebrafish. n=8-10 per group. Zebrafish morphology imaged by microscope. Figures are magnified as 32 × (whole fish) and 64 × (tail). The fluo rescence intensity was measured using image J software. Statistical analysis demonstrated the mean + SEM, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The IDO1 inhibitor 1-MT (1-methyl-tryptophan, Selleck) was solubled in dimethyl sulfoxide (DMSO) and diluted to 50 μM with filtered egg water.

Techniques: Inhibition, Immunofluorescence, Staining, Cell Culture, Imaging, Fluorescence, Microscopy, Software

Journal: Free radical biology & medicine

Article Title: Indoleamine 2, 3-dioxygenase 1 aggravates acetaminophen-induced acute liver failure by triggering excess nitroxidative stress and iron accumulation.

doi: 10.1016/j.freeradbiomed.2021.07.008

Figure Lengend Snippet: Fig. 6. IDO1 deficiency reduced iron accumulation via suppressing Tf-TfR axis. (A) Iron accumulation in different groups. (B) Immunoblotting results showed that comparing with APAP-treated WT mice, APAP-treated IDO1−/−mice inhibited Tf and TfR expression obviously. GAPDH served as a loading control. Quantitative analysis in right panel. (C, D) Immunofluorescence staining of Tf and TfR in liver tissue of both WT mice and IDO1−/−mice. Quantitative analysis in right panel respectively. The density of protein and the fluorescence intensity were measured using image J software. Statistical analysis showed the mean + SEM, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The IDO1 inhibitor 1-MT (1-methyl-tryptophan, Selleck) was solubled in dimethyl sulfoxide (DMSO) and diluted to 50 μM with filtered egg water.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Fluorescence, Software

Journal: Free radical biology & medicine

Article Title: Indoleamine 2, 3-dioxygenase 1 aggravates acetaminophen-induced acute liver failure by triggering excess nitroxidative stress and iron accumulation.

doi: 10.1016/j.freeradbiomed.2021.07.008

Figure Lengend Snippet: Fig. 7. Exhaustion of macrophages alleviated liver injury by reducing iron accumulation in hepatocytes. (A) Relative number of cells expression of CD11b and CD71 were showed by Flow cytometry. (B) Representative Immunofluorescence staining of FPN1 in RAW264.7 macrophages treated with APAP (5 mM) for 8 h. (C) Western blot showed the expression of TfR, FPN1 and IDO1 protein after 24 h of incubation with Erastin (40 μM or 80 μM) in RAW264.7 cells. (D) Representative Immunofluorescence staining of F4/80 (green) expression in mice liver. Nuclei were stained with DAPI (blue). (E, F) The serum levels of inflammatory IL-6 and MCP- 1 were detected. (G–I) Comparison of liver tissue homogenate of iron concentration, MDA and SOD level of different indicated groups were determined. (J) Immunoblotting results showed that comparing with APAP-treated WT mice, Gdcl3 treated mice inhibited TfR expression obviously. GAPDH served as a loading control. The density of protein and the fluorescence intensity were measured using image J software. Statistical analysis showed the mean + SEM, *p<0.05, **p<0.01, ***p<0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The IDO1 inhibitor 1-MT (1-methyl-tryptophan, Selleck) was solubled in dimethyl sulfoxide (DMSO) and diluted to 50 μM with filtered egg water.

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Staining, Western Blot, Incubation, Comparison, Concentration Assay, Control, Fluorescence, Software

Journal: Mucosal immunology

Article Title: Tryptophan Metabolite Activation of the Aryl Hydrocarbon Receptor Regulates IL10 Receptor Expression on Intestinal Epithelia

doi: 10.1038/mi.2016.133

Figure Lengend Snippet: (A) qPCR of il10r1 and cyp1a1 transcript levels in T84 IEC in response to AHR ligands. Confluent monolayers of T84 cells were treated with Kyn (100 μM), FICZ (250 nM), or TCDD (30 nM) for 6 h. Error bars represent the S.E.M. of five replicates. (B) Luciferase-based assay of IL10R1 promoter activity in response to AHR ligands FICZ and Kyn. IFN- γ served as a positive control. Caco-2 IECs were transfected with pGL3 containing the IL10R1 promoter region or empty pGL3 vector as control, treated for 12h with 100 μM Kyn, 250 μM FICZ, or 10 ng/mL IFN- γ and luciferase activity measured, n=3 (***p<0.001). (C) Cell surface ELISA of IL10R1 expression in shControl, shRNA ARNT knockdown, and AHR inhibitor treated Caco-2 IEC. Confluent monolayers of Caco-2 IEC were treated with 100 μM Kyn, 250 nM FICZ, or 30 nM TCDD for 24h. Error bars represent the S.E.M. of triplicate samples, *p<0.05, **p<0.01. (D) Western blot analysis of IL10R1 levels in shRNA AHR knockdown Caco-2 IEC. Confluent monolayers of Caco-2 cells containing a non-template control (shNTC) or shRNA specific for AHR were treated with FICZ or Kyn for 24 h. (E) Densitometry of the western blot analysis in . Relative density of the IL10R1 bands is normalized to β-actin. (F) Western blot analysis of IL10R1 levels in shRNA ARNT knockdown T84 IEC. Confluent monolayers of T84 cells containing a non-template control (shNTC) or shRNA specific for ARNT were treated with Kyn, FICZ, or TCDD for 24 h. (G) Densitometry of the western blot analysis in . Relative density of the IL10R1 bands is normalized to β-actin.

Article Snippet: Where indicated, cells were treated with the AHR inhibitor CH223191 (Sigma, 10 μM), the IDO1 inhibitor 1-methyl-DL-tryptophan (1-MT, Sigma), recombinant human IFN- γ (10 ng/mL, R&D), or the AHR agonists FICZ (6-Formylindolo(3,2-b)carbazole; 100 nM – 1 μM; Enzo), Kyn (Kynurenine, Sigma, 100 μM), or TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, Sigma, 30 nM) in serum free media.

Techniques: Luciferase, Activity Assay, Positive Control, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Expressing, shRNA, Western Blot