Isotype control

For KC depletion, mice were intraperitoneally (IP) injected with a single dose of 500 ng of DT (Sigma) or PBS for control mice. For TNF and IL-1 blocking experiments, 20 mg/kg of Anti-TNF-blocking antibody (Bio X Cell), 100 mg/kg of Anakinra or 20 mg/kg of isotype control (Bio X Cell) were given to mice intraperitoneal concomitant to DT injection. For EdU (Thermo Fisher) experiment, mice were IP injected with 1mg/mouse of EdU 4 h before sacrifice. For DDL1/4 blocking experiments, 1 mg of of DLL1 (BioXcell) and 1 mg of DLL4 (BioXCell) or 2 mg of polyclonal Armenian hamster IgG isotype (BioXCell) were injected 24 h before DT injection. For CSF1R blocking experiments, 100 mg/kg of PLX3397 was given by gavage 24 h and 48 h after DT injection.

Sepsis was induced by CLP^{58 (link)} and compared to sham-operated controls. Briefly, mice were anesthetized with chloral hydrate (400 mg/kg body weight) and, through a midline celiotomy, the cecum was ligated distal to the ileocecal valve and punctured with an 18-gauge needle to allow feces to enter the peritoneal cavity. A control group was sham-operated. The mice were intravenously administered with either saline or other agents, including A438079 (80 mg/kg, P2RX₇ antagonist, Tocris Bioscience, Bristol, UK), anti-IL-1β mAb (100 μg/mouse, AB-401-NA, R&D Systems, Minneapolis, MN, USA) or anti-CXCL1 mAb (50 μg/mouse, AB-401-NA, R&D Systems), anti-CX3CL1 mAb (15 μg/mouse, AB-401-NA, R&D Systems), anti-CCL2/JE/MCP-1 mAb (10 mg/kg, AF-479-NA, R&D Systems) and anti-CXCL7/Thymus Chemokine-1 mAb (50 mg/kg, AF793, R&D Systems), at 1 h after CLP surgery. Normal Goat IgG (R&D Systems), Rat IgG2A isotype control (R&D Systems) were used as isotype control and administered using the same dosing schedule. For neutrophil depletion, mice were treated with intraperitoneal injection of 250 μg anti-granulocyte receptor-1 (Gr-1) mAb RB6-8C5 (BioXCell, West Lebanon, NH, USA) or an isotype control (BioXCell) 24 h prior to CLP surgery. Assessments were made by two independent observers who were blind to genotype and treatment status.