Gsk484

Conditionally immortalized mouse and human podocytes and human glomerular endothelial cells (hGENCs) were grown on 10 cm² cell culture plates at 33 °C in the presence of interferon γ (10 U/mL), which enhances expression of the thermosensitive T antigen. These conditions are optimum for cells to proliferate and remain undifferentiated. To induce differentiation, thermoswitching was performed at 37 °C in the absence of interferon γ and the addition of 100 ng/mL vascular endothelial growth factor (VEGF). Experiments were performed after 7 days of differentiation for hGENCs and 14 days of differentiation for mouse and human podocytes. Mouse and human primary glomerular endothelial cells were obtained from cell biologicals, and 2.2 × 10⁶ cells were cultured onto 0.02% gelatin-coated plates at 37 °C in (M-6115 and H-6115 media from cell biologicals). All the cell lines tested negative for mycoplasma. In some experiments, cultured cells were treated with 25 mM glucose (SIGMA, G8769) and/or 10,000 neutrophils, 10 µM GSK484 (Cayman, 17488) for 24 h. Cells cultured under normal glucose (5 mM) or 25 mM mannitol were used as controls.

Sepsis was induced by CLP. Briefly, the peritoneal cavity was opened under inhaled isoflurane anesthesia. The cecum was eviscerated, ligated below the ileocecal valve using a 5-0 silk suture at 75% from the tip, and punctured through with a 21-gauge needle. Sham-operated animals were handled in the similar manner, except the cecum was not ligated and punctured. For the survival study, mice were monitored for 7 days. For mechanistic studies, mice were sacrificed at their respective time point. For inhibitor analysis, PAD4 inhibitor GSK484 (40 mg/kg) (Cayman Chemical, 17488), PAD2 inhibitor AFM32a (20 mg/kg), which was synthesized as previously described (65 (link)), or DMSO alone was i.p. injected 1 hour after CLP.

To induce peritonitis, mice were injected intra-peritoneally with either 0.5mg Zymosan-A (Sigma) in PBS, 0.25mg of Fluorescein labeled Zymosan-A (Invitrogen), or PBS alone and samples were isolated 2-72 h later. To block NETosis, mice were injected i.p. with 400 μg/mouse of GSK484 (Cayman Chemical). Blocking antibodies against CXCL1 (Clone 48415, Invitrogen) or isotype control Rat IgG2a (Invitrogen) antibodies were injected i.p. 2 h after induction of peritonitis (40 μg/mouse). Following Zymosan and anti-CXCL1 treatment, omenta were isolated and cultured with mCherry E. coli (Amarh et al., 2018 (link)) or E. coli bioparticles (Invitrogen) in vitro for 5 min prior to thorough washing with RPMI-1640 (Sigma), and wholemount staining as described below.
Peritoneal exudate cells (PEC) were isolated by flushing murine peritoneal cavities with RPMI 1640 (Sigma). Murine omenta were enzymatically digested with 1mg/mL Collagenase D (Roche) for 35 min at 37°C in RPMI 1640 (Sigma) containing 1% Fetal Bovine Serum (FBS) (Sigma). Spleens were mechanically disrupted using glass slides.