Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dynamics of the IL-33/ST2 network in the progression of human colorectal adenoma to sporadic colorectal cancer

doi: 10.1007/s00262-014-1624-x

Figure Lengend Snippet: Dynamic changes in IL-33 and ST2 transcripts along the colorectal adenoma–carcinoma sequence. The quantitative real-time PCR results showed that the expression of IL-33 mRNA was significantly increased to a ~ninefold higher level in adenoma tissues (gray bar in a); it was also higher in sporadic CRC tissues (black bar in a) than in normal controls (white bar in a), but significantly lower compared with the adenomas. The expression of ST2 mRNA in adenoma tissues was increased to a ~threefold higher level and slightly increased in CRC tissues compared with normal controls (b). (Y axes in a, b are fold changes relative to normal controls; P values are derived from the Mann–Whitney test.)

Article Snippet: The following primary antibodies were used: goat antihuman IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and mouse antihuman ST2 monoclonal antibody (working dilution 1:100; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan).

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, MANN-WHITNEY

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dynamics of the IL-33/ST2 network in the progression of human colorectal adenoma to sporadic colorectal cancer

doi: 10.1007/s00262-014-1624-x

Figure Lengend Snippet: Kaplan–Meier curves of overall survival differences among patients with sporadic CRC. The Kaplan–Meier analysis shows that the expression levels of IL-33 (a) and ST2 (b) do not predicate the overall survival time in patients with sporadic CRC (both P values determined by the log-rank test)

Article Snippet: The following primary antibodies were used: goat antihuman IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and mouse antihuman ST2 monoclonal antibody (working dilution 1:100; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan).

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dynamics of the IL-33/ST2 network in the progression of human colorectal adenoma to sporadic colorectal cancer

doi: 10.1007/s00262-014-1624-x

Figure Lengend Snippet: Photograph presentations of IL-33 and its receptor, ST2, in the tumor stroma and the adenomatous/cancerous epithelium. Immunohistochemical (IHC) results show that IL-33 immunoreactivity was not observed in the normal epithelium, and a low density of IL-33-positive cells was found in the lamina propria in the control (a). In both the adenoma and CRC, IL-33 immunoreactivity was frequently observed in tumor-associated microvessels (arrow in b, c) and adenomatous/cancerous epithelium (arrow in b for adenoma and inserted image in 3C for CRC). ST2 immunoreactivity was observed in both the epithelium (arrow head) and microvessels (arrow) in all three groups (d for control, e for adenoma and f for CRC). (a–e IHC, counterstained with hematoxylin, original magnification 200×.)

Article Snippet: The following primary antibodies were used: goat antihuman IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and mouse antihuman ST2 monoclonal antibody (working dilution 1:100; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan).

Techniques: Immunohistochemical staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dynamics of the IL-33/ST2 network in the progression of human colorectal adenoma to sporadic colorectal cancer

doi: 10.1007/s00262-014-1624-x

Figure Lengend Snippet: Density grading scores of IL-33- and ST2-positive cells in adenomas and CRC tissues. The semi-quantitative results showed that the density of IL-33-positive cells in the adenomatous epithelium (gray bar in a) was higher than in the controls. The density of IL-33-positive cells was also higher in the CRC epithelium (black bar in a) compared with the controls, but lower than the adenomatous epithelium (Fig. a). The density of IL-33-positive stromal cells was increased in the adenoma stroma, with a smaller increase in magnitude of density in the CRC stroma (b). The density of ST2-positive cells in the adenomatous epithelium was non-statistically increased and was unchanged in the CRC cancerous epithelium (c). The density of ST2-positive cells in the stroma showed a gradually increasing trend from the control to adenoma to CRC, but these differences were not statistically significant (d). To examine the expression of IL-33/ST2 in endothelial cells, the number of IL-33-positive tumors associated with microvessel density (MVD) and the number of ST2-positive tumors associated with MVD were counted. The results show that the IL-33-positive MVD was significantly increased in the adenoma stroma and non-statistically increased in the CRC tumor stroma compared with normal lamina propria (e). The number of ST2-positive microvessels was greatly increased in both the adenoma stroma and the CRC tumor stroma compared with the normal lamina propria (f). (HPF high-power field.)

Article Snippet: The following primary antibodies were used: goat antihuman IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and mouse antihuman ST2 monoclonal antibody (working dilution 1:100; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan).

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dynamics of the IL-33/ST2 network in the progression of human colorectal adenoma to sporadic colorectal cancer

doi: 10.1007/s00262-014-1624-x

Figure Lengend Snippet: Phenotypic characterization of IL-33-expressing cells in the adenoma and CRC tumor stromas. Double IHC with IL-33/CD34 and IL-33/SMA-alpha revealed that an increase in IL-33-positive cells (red) in the adenoma and CRC tumor stromas was frequently co-localized with CD34-labeled microvessels (blue) (b, c) and SMA-alpha-labeled myofibroblasts (blue) (e, f) compared with the normal lamina propria (a, d). (a–f double IHC, original magnification 400×; counterstaining was not applied.)

Article Snippet: The following primary antibodies were used: goat antihuman IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and mouse antihuman ST2 monoclonal antibody (working dilution 1:100; Medical & Biological Laboratories Co. Ltd., Nagoya, Japan).

Techniques: Expressing, Labeling

Journal: Cellular and Molecular Immunology

Article Title: Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells

doi: 10.1038/cmi.2016.63

Figure Lengend Snippet: IL-33 expression in acute experimental and clinical liver fibrosis. (a) The hepatic IL-33 expression following bile-duct ligation (BDL) was assessed in C57BL/6 mice at several time points by real-time PCR and western blot, and (b) IL-33 serum levels were determined by ELISA. Mean values±s.e.m. are given of two independent studies with groups of eight mice (*P<0.05, **P <0.01, ***P <0.001). (c) IL-33 in human liver tissue homogenates (ELISA) after partial hepatectomy of early-stage hepatocellular carcinoma with fibrosis or controls with hepatic hemangioma. The data are expressed as the median and range (0, 25, 50, 75 and 100%) from 24 fibrotic and 20 normal liver tissues (***P <0.001). (d) Cellular expression of IL-33 in fibrotic human livers was identified by immunofluorescence (IF) using FITC-labeled anti huIL-33 antibody, PE-labeled CK-19 (cholangiocytes), PE-labeled GFAP (HSC) or PE-labeled albumin (hepatocytes). Nuclei were counterstained with DAPI (magnification × 400, scale bar, 50 μm).

Article Snippet: In preliminary trials, mice were given an intraperitoneal injection of recombinant mouse IL-33 (1, 5 or 10 μg per day, R&D Systems, Abingdon, UK) 3 days before BDL, and 5 μg was finally used in the experiments.

Techniques: Expressing, Ligation, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling

Journal: Cellular and Molecular Immunology

Article Title: Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells

doi: 10.1038/cmi.2016.63

Figure Lengend Snippet: BDL-induced liver injury and fibrosis are reduced in the absence of ST2. Liver injury following bile-duct ligation (BDL) was analyzed in C57BL/6 and ST2-deficient (KO) mice. (a) Macro- and microphotographs demonstrate attenuated liver inflammation, necrosis and fibrosis in ST2-KO mice (H&E and Masson staining, original magnification × 200). (b) Serum alanine aminotransferase and aspartate aminotransferase at 0, 1, 3, 10 and 21 days after BDL. (c) Inflammation was assessed in liver homogenates of C57BL/6 and IL-33/ST2-KO mice at 0, 1, 3, 10 and 21 days after BDL. IL-1β, KC and thymic stromal lymphopoietin were reduced in ST2-KO mice compared with C57BL/6 mice. (d) Hepatic collagen expression was assessed with a Sircol assay and real-time PCR for collagen 1a1. The data are expressed as median values±s.e.m. (n=6–8 mice/group) and are representative of three independent experiments (*P<0.05, **P<0.01, ***P<0.001).

Article Snippet: In preliminary trials, mice were given an intraperitoneal injection of recombinant mouse IL-33 (1, 5 or 10 μg per day, R&D Systems, Abingdon, UK) 3 days before BDL, and 5 μg was finally used in the experiments.

Techniques: Ligation, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Immunology

Article Title: Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells

doi: 10.1038/cmi.2016.63

Figure Lengend Snippet: Recombinant IL-33 exacerbates inflammation in C57BL/6 mice. Recombinant mouse IL-33 was injected 3 days before BDL (intraperitoneally at 5 μg per day) into C57BL/6 mice. (a) Serum alanine aminotransferase and aspartate aminotransferase levels from WT BL6 mice without or with rmIL-33 (0 μg, 1 μg, 5 μg and 10 μg) at 1 day. The data are expressed as the mean values±s.e.m. (n=6–8 mice/group; (a) values are significantly different from the BL6 sham group; (b) not significantly different from the 5 μg IL-33 group). (b and c) Increased liver injury and inflammation, as assessed by hematoxylin and eosin staining, myeloperoxidase and Suzuki score. (d) Hepatic IL-1β, thymic stromal lymphopoietin and granulocyte-macrophage colony stimulating factor were increased. The data are expressed as the mean values±s.e.m. (n=6–8 mice/group) and are representative of three independent experiments (*P<0.05; **P<0.01; ns, not significant).

Article Snippet: In preliminary trials, mice were given an intraperitoneal injection of recombinant mouse IL-33 (1, 5 or 10 μg per day, R&D Systems, Abingdon, UK) 3 days before BDL, and 5 μg was finally used in the experiments.

Techniques: Recombinant, Injection, Staining

Journal: Cellular and Molecular Immunology

Article Title: Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells

doi: 10.1038/cmi.2016.63

Figure Lengend Snippet: IL-33 activates hepatic stellate cells (HSCs) via mitogen-activated protein kinases signaling. Mouse HSCs were isolated from naive C57BL/6 and ST2-KO mice and activated with rmuIL-33 in vitro (0, 1, 10, 50 or 100 ng/ml). IL-6, TGF-β, α-SMA RNA transcription and soluble collagen in the supernatant were increased at 24 h (a–d). HSCs expressed increased phosphorylated JNK, ERK or p38 on activation by rmuIL-33 at 24 h, which was ST2-dependent. Levels of JNK, ERK or p38 phosphorylation were quantified by ImageJ software (e). The JNK inhibitor SP600125, ERK/MEK1 inhibitor PD98059, or p38 inhibitor SB203580 were given 1 h before rmIL-33 at 100 ng/ml and inhibited collagen production, as measured by Sircol assay (f). Mean values±s.e.m. of a representative study of three independent experiments are shown; comparisons between subgroups were performed by one-way ANOVA followed by a Newman–Keuls posttest: *P<0.05, **P<0.01, ***P<0.001 (compared with cells cultured in medium).

Article Snippet: In preliminary trials, mice were given an intraperitoneal injection of recombinant mouse IL-33 (1, 5 or 10 μg per day, R&D Systems, Abingdon, UK) 3 days before BDL, and 5 μg was finally used in the experiments.

Techniques: Isolation, In Vitro, Activation Assay, Software, Cell Culture

Journal: JCI Insight

Article Title: Sex differences in IL-17 contribute to chronicity in male versus female urinary tract infection

doi: 10.1172/jci.insight.122998

Figure Lengend Snippet: (A–C) Female and male mice were infected with 1 × 107 CFU of UPEC UTI89-RFP-kanR and bladders analyzed at 24 hours PI. Graphs show (A) IL-33 protein levels in homogenized bladder, (B) IL-4Rα expression on bladder resident macrophages (Mϕ), and (C) the number of ILCs (CD90+CD3–CD4–NK1.1–MHC II–CD11b–) in bladders. (D–F) Female mice were implanted with empty tubing (Mock) or slow-release tubing containing testosterone (T tube) and allowed to recover 1 week before infection with 1 × 107 CFU UPEC strain UTI89-RFP-kanR. Graphs show (D) IL-33 protein levels in homogenized bladder tissue, (E) IL-4Rα expression on bladder resident macrophages, and (F) the number of ILCs in bladders. Data are pooled from 2–3 experiments, n = 4–5 mice/group in each experiment. Each dot is 1 mouse, red dots depict female mice and blue dots are male mice, and lines are medians. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann-Whitney test. Analyses in this figure were corrected for multiple testing by the Holm–Bonferroni method; all P < 0.05 had q < 0.05.

Article Snippet: For IL-33 neutralization, 3.6 μg/mouse α–IL-33 (catalog number AF3626, BioTechne/R&D Systems) or 3.6 μg/mouse polyclonal goat IgG isotype control (catalog number AB-108-C, BioTechne/R&D Systems) was delivered intraperitoneally in 100 μL PBS ( 44 ).

Techniques: Infection, Expressing, MANN-WHITNEY