Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Inhaled house dust programs pulmonary dendritic cells to promote type 2 T-cell responses by an indirect mechanism

doi: 10.1152/ajplung.00256.2015

Figure Lengend Snippet: HDE-mediated conditioning of cDCs does not involve IL-25, IL-33, or thymic stromal lymphopoietin (TSLP). A: FL-cDCs prepared from C57BL/6 mice were treated with BALF-HDE from WT (open bars) or Il33−/− mice (solid bars) for 24 h and then cultured with naïve CD4+ OT-II cells in the presence of OVA peptide to induce Th cell differentiation. Bars represent the means ± SD of duplicate wells. Data are from 1 experiment, representative of 2 experiments. B and C: FL-cDCs were treated with BALF-HDE in the presence of neutralizing anti-IL-25 (B, solid bars), anti-TSLP (C, solid bars), or isotype control antibodies (B and C, open bars) for 24 h and then cultured with naïve CD4+ OT-II cells in the presence of OVA peptide to induce Th cell differentiation. Bars represent the means ± SD of duplicate wells. Data are from 1 experiment, representative of 2 experiments. **P < 0.01.

Article Snippet: In some experiments, 10 μg/ml of neutralizing polyclonal anti-mouse TSLP (R&D Systems Minneapolis, MN) ( 36 ), neutralizing monoclonal anti-mouse IL-17E/IL-25 (Clone 50104, R&D Systems), or isotype control antibodies were added to DC cultures during BALF treatment.

Techniques: Cell Culture, Cell Differentiation

Journal: Frontiers in immunology

Article Title: IL25 Enhanced Colitis-Associated Tumorigenesis in Mice by Upregulating Transcription Factor GLI1.

doi: 10.3389/fimmu.2022.837262

Figure Lengend Snippet: FIGURE 1 | Overexpression of IL25 was found in CRC patients and predicts a poor prognosis. (A) Immunohistochemistry (IHC) staining of IL25 was performed in a tissue microarray consisting of 74 CRC tumor tissues and adjacent colon tissues (left). Statistical analysis of IL25 staining in adjacent specimens and CRC specimens (right). (B) Protein levels of IL25 were detected by Western blotting in normal intestinal cells (CCD841) and CRC cell lines (left). The right panel showed the quantitative analysis of the gray scan. The ImageJ software was used for gray scanning. (C) Representative images of IL25 IHC staining at different clinical stages (up). Correlation between IL25 expression and various clinical stages (down). (D) Overall survival curves of 49 CRC patients in correlation with intra-tumor IL25 IHC-scores. High IL25 expression was considered IHC-Score >6. The patients with CRC were divided into 2 groups according to the intra- tumor IL25 IHC-score: low group (n = 34), high group (n = 15). (E) Representative images of IL25 IHC staining from WT colon and AOM/DSS induced tumors on weeks 10 and 16 (down). Statistical analysis of IL25 staining in con colon, adjacent tissues, and AOM/DSS-induced CRC tissues (up). Data present as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For IL25 immunohistochemistry, slides of various tissues were blocked with goat serum for 1 h. Subsequently, the slides were incubated with the following primary antibodies: IL25 (1:200; Novus Biologicals, NB100-56541) and LGR5 (1:100; Abcam, ab75732) antibody overnight at 4°C following incubation with HRPconjugated secondary antibody for 1 h at room temperature and then stained with the DAB Horseradish Peroxidase Color Development Kit.

Techniques: Over Expression, Immunohistochemistry, Microarray, Staining, Western Blot, Software, Expressing

Journal: Frontiers in immunology

Article Title: IL25 Enhanced Colitis-Associated Tumorigenesis in Mice by Upregulating Transcription Factor GLI1.

doi: 10.3389/fimmu.2022.837262

Figure Lengend Snippet: FIGURE 2 | Genetic deletion of IL25 inhibited the progression of the Colitis-Associated Cancer (CAC) Model. IL25KO or WT control mice were given an intraperitoneal injection of AOM on day 1, 2.5% DSS in drinking water for 7 days starting on days 7, 28, and 42, and euthanized on days 70 and 112. (A) Bodyweight change during colitis-associated colorectal cancer with AOM/DSS as a percentage of initial weight. (B) Overall survival curves of WT and IL25KO mice. (C) Representative images of colonic tumors from WT and IL25KO mice in 10 weeks. (D) Total number and size of tumors along the colon in WT (n = 5) and IL25KO (n = 5). (E) Colon length in mice treated with the indicated treatment in 10 weeks. (F) Effect of oxaliplatin on WT and IL25KO AOM-DSS-induced CRC mouse models. The colon was removed, cut lengthwise, washed with PBS, and digitally photographed. (G) Size of individual tumors along the colon in WT treated with vehicle (n = 4) or oxaliplatin (n = 4) and IL25KO treated with vehicle (n = 7) or oxaliplatin (n = 7). (H) Total number and size of tumors. (I) Colon length in mice treated with the indicated treatment on 16 weeks. (J) Representative immunofluorescent stains for TUNEL in colonic sections from WT and IL25KO treated with vehicle or oxaliplatin (left). Statistical analysis of Tunel staining in WT and IL25KO tumors (right). Data present as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For IL25 immunohistochemistry, slides of various tissues were blocked with goat serum for 1 h. Subsequently, the slides were incubated with the following primary antibodies: IL25 (1:200; Novus Biologicals, NB100-56541) and LGR5 (1:100; Abcam, ab75732) antibody overnight at 4°C following incubation with HRPconjugated secondary antibody for 1 h at room temperature and then stained with the DAB Horseradish Peroxidase Color Development Kit.

Techniques: Control, Injection, TUNEL Assay, Staining

Journal: Frontiers in immunology

Article Title: IL25 Enhanced Colitis-Associated Tumorigenesis in Mice by Upregulating Transcription Factor GLI1.

doi: 10.3389/fimmu.2022.837262

Figure Lengend Snippet: FIGURE 3 | IL25 promoted the stemness of CRC cells. (A) The expression levels of CSC markers, namely, CD133 and LGR5, were examined in HT-29 and SW620 cells treated with recombinant IL25 in a concentration-dependent manner by RT-PCR. (B, C) The expression levels of CD133 and LGR5 were examined in HT-29 and SW620 cells treated with recombinant IL25 in a concentration and time-dependent manner by Western blotting. (D) Frequency (down) and representative day-7 images (up) of human colon adenomatous organoids treated with/without IL25 (50 ng/ml). (E) Sphere formation analysis of HT-29 and SW620 cells treated with recombinant IL25 in a concentration-dependent manner. Representative images (up) and the mean numbers and sphere size (down) of spheres are shown. Scale bar, 200 mm. Data present as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For IL25 immunohistochemistry, slides of various tissues were blocked with goat serum for 1 h. Subsequently, the slides were incubated with the following primary antibodies: IL25 (1:200; Novus Biologicals, NB100-56541) and LGR5 (1:100; Abcam, ab75732) antibody overnight at 4°C following incubation with HRPconjugated secondary antibody for 1 h at room temperature and then stained with the DAB Horseradish Peroxidase Color Development Kit.

Techniques: Expressing, Recombinant, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in immunology

Article Title: IL25 Enhanced Colitis-Associated Tumorigenesis in Mice by Upregulating Transcription Factor GLI1.

doi: 10.3389/fimmu.2022.837262

Figure Lengend Snippet: FIGURE 4 | IL25 Deficiency Induced loss of CRC stemness. (A) Representative images of LGR5 IHC staining (left). Statistical analysis of LGR5 staining in WT and IL25KO tumors (right). (B) Stem cell markers CD133 and LGR5 expression were detected by multiplexed fluorescence staining in the WT and IL25KO AOM- DSS-induced CRC mouse models. The representative images show the expression of LGR5 (red), DAPI (blue), CD133 (green). (C) The expression levels of stem cells markers were examined in WT and IL25KO tumors by RT-PCR. (D) The expression levels of CSC markers, namely, DCLK1, ALDH1, and CD44, were examined in tumor tissues by Western blotting. (E) Western blotting of Stem cell markers levels in LoVo cells following IL25 silencing. (F) Frequency (right) and representative day-7 images (left) of adenomatous organoids from WT and IL25KO mice treated with/without IL25 (50 ng/ml). (G) Sphere formation analysis of LoVo cells following IL25 silencing. Representative images (left) and the mean numbers and sphere size (right) of spheres are shown. Data present as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For IL25 immunohistochemistry, slides of various tissues were blocked with goat serum for 1 h. Subsequently, the slides were incubated with the following primary antibodies: IL25 (1:200; Novus Biologicals, NB100-56541) and LGR5 (1:100; Abcam, ab75732) antibody overnight at 4°C following incubation with HRPconjugated secondary antibody for 1 h at room temperature and then stained with the DAB Horseradish Peroxidase Color Development Kit.

Techniques: Immunohistochemistry, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in immunology

Article Title: IL25 Enhanced Colitis-Associated Tumorigenesis in Mice by Upregulating Transcription Factor GLI1.

doi: 10.3389/fimmu.2022.837262

Figure Lengend Snippet: FIGURE 5 | IL25 promoted stemness via the Hedgehog signaling pathway in CRC cancer. (A) GSEA for Hedgehog signaling pathway (nominal p < 0.05) in IL25 high expression colorectal cancer compared with IL25 low expression colorectal cancer from GSE41258. (B) The expression levels of Hedgehog signaling genes were examined in HT-29 and SW620 cells treated with recombinant IL25 in a concentration-dependent manner by RT-PCR. (C) The expression levels of Hedgehog signaling genes were examined in WT and IL25KO AOM/DSS-induced CRC cancer tissues by RT-PCR. (D) GLI1 expression was detected by immunofluorescence staining in HT- 29 cells treated with or without 50 ng/ml IL25. (E) GLI1 and CD133 expression were detected by multiplexed fluorescence staining in the WT and IL25KO AOM/DSS- induced tumor tissues. Representative images show the expression of GLI1 (red), DAPI (blue), and CD133 (green). (F) Western blotting of Hedgehog signaling genes in LoVo cells following IL25 silencing. (G) Western blotting of GLI1 and CD133 in HT-29 and SW620 cells treated with SMO inhibitor Vismodegib and GLI1 inhibitor GANT- 58 following IL25 treatment. (H) Sphere formation analysis of SW620 cells treated with SMO inhibitor Vismodegib and GLI1 inhibitor GANT-58 following IL25 treatment. Representative images (left) and the mean numbers and sphere size (right) of spheres are shown. Data present as mean ± SEM; *p < 0.05, ***p < 0.001.

Article Snippet: For IL25 immunohistochemistry, slides of various tissues were blocked with goat serum for 1 h. Subsequently, the slides were incubated with the following primary antibodies: IL25 (1:200; Novus Biologicals, NB100-56541) and LGR5 (1:100; Abcam, ab75732) antibody overnight at 4°C following incubation with HRPconjugated secondary antibody for 1 h at room temperature and then stained with the DAB Horseradish Peroxidase Color Development Kit.

Techniques: Expressing, Recombinant, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Western Blot

Journal: Frontiers in immunology

Article Title: IL25 Enhanced Colitis-Associated Tumorigenesis in Mice by Upregulating Transcription Factor GLI1.

doi: 10.3389/fimmu.2022.837262

Figure Lengend Snippet: FIGURE 6 | IL25 upregulated GLI1 by inhibiting p-AMPK. (A) HT-29 cells were treated with cycloheximide (CHX, 50 mg/ml) for the indicated time, and cell lysates were analyzed by Western blotting with the indicated antibodies. (B) Western blotting of p-AMPK and AMPK in the WT and IL25KO AOM/DSS-induced tumor tissue. (C) The expression levels of GLI1, p-AMPK, and AMPK were examined in HT-29 and SW620 cells treated with recombinant IL25 in a time-dependent manner by Western blotting. (D, E) Western blotting of GLI1, p-AMPK, and AMPK in SW620 cells treated with AMPK activator A769662 and Metformin following IL25 treatment. (F) GLI1 expression was detected by immunofluorescence staining in SW620 cells treated with AMPK activator Metformin following IL25 treatment. (G) SW620 cells were treated with 10 mM MG132 and then incubated with or without 50 ng/ml recombinant IL25 and 1 mM Metformin, then immunoprecipitated with GLI1 antibody. GLI1 ubiquitination was determined using an anti-ubiquitin antibody. IP, immunoprecipitation. (H) Sphere formation analysis of SW620 cells treated with AMPK activator Metformin following IL25 treatment. Representative images (left) and the mean numbers and sphere size (right) of spheres are shown. Data present as mean ± SEM; *p <0.05, **p <0.01, ***p <0.001.

Article Snippet: For IL25 immunohistochemistry, slides of various tissues were blocked with goat serum for 1 h. Subsequently, the slides were incubated with the following primary antibodies: IL25 (1:200; Novus Biologicals, NB100-56541) and LGR5 (1:100; Abcam, ab75732) antibody overnight at 4°C following incubation with HRPconjugated secondary antibody for 1 h at room temperature and then stained with the DAB Horseradish Peroxidase Color Development Kit.

Techniques: Western Blot, Expressing, Recombinant, Staining, Incubation, Immunoprecipitation, Ubiquitin Proteomics

Journal: Frontiers in immunology

Article Title: IL25 Enhanced Colitis-Associated Tumorigenesis in Mice by Upregulating Transcription Factor GLI1.

doi: 10.3389/fimmu.2022.837262

Figure Lengend Snippet: FIGURE 7 | Proposed model for the roles and functions of IL25 in promoting CRC stemness. IL25 can inhibit p-AMPK and lead to GLI1 accumulation. It also stimulates SHH secretion by the means that minus times minus equals plus, thereby GLI1 binding with SMO. In nuclear, GLI1 can promote stem cell markers and ABC transporters expression. Therefore, CRC cells gain the ability of stemness and oxaliplatin resistance.

Article Snippet: For IL25 immunohistochemistry, slides of various tissues were blocked with goat serum for 1 h. Subsequently, the slides were incubated with the following primary antibodies: IL25 (1:200; Novus Biologicals, NB100-56541) and LGR5 (1:100; Abcam, ab75732) antibody overnight at 4°C following incubation with HRPconjugated secondary antibody for 1 h at room temperature and then stained with the DAB Horseradish Peroxidase Color Development Kit.

Techniques: Binding Assay, Expressing