Journal: International Journal of Molecular Sciences

Article Title: Natterin-Induced Neutrophilia Is Dependent on cGAS/STING Activation via Type I IFN Signaling Pathway

doi: 10.3390/ijms23073600

Figure Lengend Snippet: Mitochondrial dysfunction and IL-33 induced in Natterin response. Natterin (1 μg) diluted in PBS was injected i.p. in BL6 WT mice previously treated for 1 h with i.p. injection of cyclosporin A at 10 μM ( WT CycA_Natterin) or DPI at 100 μM ( WT DPI_Natterin) ( A ). As a negative control, mice were injected i.p. with PBS ( WT _PBS). As a positive control, mice were only injected with Natterin ( WT _Natterin). Two hours after injection, mice were killed and the peritoneal cavities were washed to obtain exudates. Peritoneal exudate cells were harvested and the number of neutrophils related to total cell number was evaluated in cytospin slides stained with the Diff-Quick staining kit. Each bar represents the mean ± SEM of 3–5 animals/group. * p < 0.05 compared with WT _PBS group and # p < 0.05 compared with WT _Natterin group. Concentrated supernatant of peritoneal exudates from WT _PBS or WT _Natterin groups of mice were analyzed for the content of double-stranded DNA using Quant-iTPicoGreen dsDNA reagent ( B ). Proteins present in concentrated supernatant or cytoplasmic and nuclear proteins ( C ) collected 2 h after Natterin injection were analyzed using the iBind TM Flex Western System with goat anti-mouse IL-33 (processed form: 18 to 20 kDa) followed by the secondary antibody anti-goat IgG-HRP. The β-tubulin was used as housekeeping protein. The immune complex was revealed by enhanced chemiluminescence detection system.

Article Snippet: Specific proteins were detected according to Grund et al. [ ] using the goat anti-mouse IL-33 (processed form: 18 to 20 kDa, no. 842875 at 1 μg/mL, R&D Systems, Inc., Minneapolis, MI, USA) followed by the second antibody donkey anti-goat IgG-HRP (no. sc 2033 at 1/2000, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and anti-β-tubulin (no. IMG-5810A at 0.6 μg/mL, IMGENEX, Novus Biologicals LLC, Littleton, CO, USA) followed by secondary antibody mouse anti-rabbit IgG HRP True Blot (no. 18-8816-33 at 1/500, Rockland, Hamburg, Germany) for 3 h on the iBindTM Flex Western System apparatus using iBind Cards (Invitrogen Thermo Fisher Scientific, no. SLF2010).

Techniques: Injection, Negative Control, Positive Control, Staining, Diff-Quik, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Natterin-Induced Neutrophilia Is Dependent on cGAS/STING Activation via Type I IFN Signaling Pathway

doi: 10.3390/ijms23073600

Figure Lengend Snippet: Neutrophilic inflammation induced by Natterin requires cGAS/STING/IRF3 via type I IFN receptor. Natterin induces neutrophilic infiltration with cell activation and release of cytosolic molecules, such as DNA, succinate and ROS. cGAS/STING drives IRF3-mediated inflammation dependent on type I IFN receptor. The activation of the STING pathway requires TLR4/TRIF-dependent pathway, essential for the production of type I IFNs, which synergizes with processed IL-33 to coordinate inflammation. Our data clarify that the neutrophilic inflammation induced by Natterin an aerolysin-like toxin is the result of activation of cytosolic DNA sensors pointing to the possibility of new pharmacological tools for its control.

Article Snippet: Specific proteins were detected according to Grund et al. [ ] using the goat anti-mouse IL-33 (processed form: 18 to 20 kDa, no. 842875 at 1 μg/mL, R&D Systems, Inc., Minneapolis, MI, USA) followed by the second antibody donkey anti-goat IgG-HRP (no. sc 2033 at 1/2000, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and anti-β-tubulin (no. IMG-5810A at 0.6 μg/mL, IMGENEX, Novus Biologicals LLC, Littleton, CO, USA) followed by secondary antibody mouse anti-rabbit IgG HRP True Blot (no. 18-8816-33 at 1/500, Rockland, Hamburg, Germany) for 3 h on the iBindTM Flex Western System apparatus using iBind Cards (Invitrogen Thermo Fisher Scientific, no. SLF2010).

Techniques: Activation Assay

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