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

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: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: A Role for Primary Cilia in Aortic Valve Development and Disease

doi: 10.1002/dvdy.24524

Figure Lengend Snippet: (A) Three-dimensional reconstruction of IHC stain at postnatal day 0, shows smoothened (red), acetylated tubulin—cilia axoneme (green), and Hoechst--nuclei (blue). High magnification (right) shows smoothened (arrowhead pointing to smoothened staining) on the axoneme of the cilia indicative of active hedgehog signaling. (B) IHC of P0 aortic cusps showing Gli3 expression.

Article Snippet: The following are the antibodies and their dilutions; Acetylated Tubulin (Sigma, Cat#T6793, 1:500), Gamma Tubulin (Abcam, Cambridge, MA, USA, Cat#ab11317, 1:1000), Versican (gift from Stan Hoffman, Medical University of South Carolina, 1: 250), Collagen (gift from Stan Hoffman, Medical University of South Carolina, 1: 250), MF20 (DSHB, Iowa City, IA, USA, Concentrate, I:50), Ki67 (Abcam, Cat#ab16667, 1:250), Phospho-histone H3 (EMD Millipore, Darmstadt Germany, Cat#06-570, 1:250), Smoothened (LSBio, Seattle WA, Cat#LS-A2666, 1:250), Gli3 (Origene, Rockville MD, Cat#TA337186, 1:250).

Techniques: Staining, Expressing