Extracellular CIRP Induces Macrophage Extracellular Trap Formation Via Gasdermin D Activation

Extracellular CIRP Induces Macrophage Extracellular Trap Formation Via Gasdermin D Activation

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Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern promoting inflammation and tissue injury.During bacterial or viral infection, macrophages release DNA decorated with nuclear and cytoplasmic proteins known as macrophage extracellular traps (METs).Gasdermin D (GSDMD) is a pore-forming protein that has been involved in extracellular trap formation in neutrophils.We hypothesized that eCIRP induces MET formation by activating GSDMD.

Human monocytic cell line THP-1 cells were differentiated with Lunch boxes phorbol 12-myristate 13-acetate (PMA) and treated with recombinant murine (rm) CIRP.The MET formation was detected by three methods: time-lapse fluorescence microscopy (video imaging), colorimetry, and ELISA.Cleaved forms of GSDMD, and caspase-1 were detected by Western blotting.Treatment of THP-1 cells with rmCIRP increased MET formation as revealed by SYTOX Orange Staining assay in a time- and dose-dependent manner.

METs formed by rmCIRP stimulation were further confirmed by extracellular DNA, citrullinated histone H3, and myeloperoxidase.Treatment of THP-1 Gas Cooker cells with rmCIRP significantly increased the cleaved forms of caspase-1 and GSDMD compared to PBS-treated cells.Treatment of macrophages with caspase-1, and GSDMD inhibitors z-VAD-fmk, and disulfiram, separately, significantly decreased rmCIRP-induced MET formation.We also confirmed rmCIRP-induced MET formation using primary cells murine peritoneal macrophages.

These data clearly show that eCIRP serves as a novel inducer of MET formation through the activation of GSDMD and caspase-1.