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